Therapeutic agent for emphysema and copd

ABSTRACT

The invention described herein relates to methods of treating emphysema and COPD with a GHK tripeptide. The invention further relates to methods of determining the state of the lungs using biomarkers described herein.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of co-pending U.S. application Ser. No.14/004,781 filed Mar. 20, 2014, which is a 35 U.S.C. §371 National PhaseEntry Application of International Application No. PCT/US2012/029823,filed Mar. 20, 2012, which designates the United States, and whichclaims the benefit under 35 U.S.C. §119(e) of U.S. Provisional PatentApplication Ser. No. 61/454,566 filed Mar. 20, 2011 and U.S. ProvisionalPatent Application Ser. No. 61/498,297 filed Jun. 17, 2011, the contentsof each of which are herein incorporated by reference in theirentireties.

GOVERNMENT SUPPORT

The present application was made with Government support under GrantNumber R01 HL095388 awarded by the National Institutes of Health. TheGovernment of the United States has certain rights in the invention.

FIELD OF THE INVENTION

The invention relates to methods of treating emphysema and COPD with thetripeptide GHK. The invention further relates to methods of detectingthe presence and severity of emphysema using the expression level ofmarker genes.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Sep. 11, 2013, isnamed 701586-069653_SL and is 2,566,572 bytes in size.

BACKGROUND OF THE INVENTION

Chronic Obstructive Pulmonary Disease (COPD) is a worldwide publichealth problem and is the fourth leading cause of death in the UnitedStates (Heron et al., National Vital Statistics Reports 2009 57:1-134).COPD is characterized by irreversible airflow limitation due toobstruction in the small conducting airways and emphysematousdestruction of the gas exchanging surface of the lung. Tobacco smoke isthe major risk factor for COPD as 10-20% of smokers develop this disease(Fletcher and Peto British Medical Journal 1977 1:1645-8). Currenttheories concerning pathogenesis of COPD include an imbalance betweenprotease and anti-protease activity, induced apoptosis of alveolar wallcells through deregulation of pathways involved in oxidative stress,angiogenesis, and chronic inflammation, and aberrant tissue remodelingand repair processes that lead to the destruction of the extra cellularmatrix (ECM) in the lung. However, the etiology of the initiation andprogression of COPD remain poorly understood.

Several groups have profiled gene expression in lung tissue frompatients with and without COPD or between patients with varying levelsof airflow obstruction in order to understand differences in geneexpression related to COPD (Golpon et al., Am J Respir Cell Mol Biol2004 31:595-600; Ning et al., PNAS 2004 101:14895-14900; Bhattacharya etal., Am J Respir Cell Mol Biol 2009 40:359-367; Spira et al., Am JRespir Cell Mol Biol 2004 31:601-610; Wang et al., Am J Respir Cell MolBiol 2008 177:402-411). Although these studies have provided an initiallook into the COPD transcriptome, their results have limited value indiagnosing COPD because they primarily relied on the use of lungfunction testing to define the presence or degree of COPD. Lung functiontesting cannot distinguish between obstruction in the small airways andemphysematous destruction of the lung parenchyma nor provide informationabout regional differences in disease severity.

Accordingly, a need exists for novel therapies for the treatment ofemphysema and COPD as well as improved diagnostics for the presence andseverity of emphysema in a subject.

SUMMARY OF THE INVENTION

Embodiments of the present invention are based on the discovery that asemphysematous destruction of lung tissue increases, the expression ofcertain genes relating to inflammatory processes is upregulated whilethe expression of certain genes associated with tissue remodeling andthe TGF-β signaling pathway is downregulated. Another aspect relates tothe discovery that the tripeptide Glycine-Histidine-Lysine (GHK) iscapable of inducing the expression of the same genes which are normallydownregulated during emphysematous destruction.

As described herein, the inventors have demonstrated that GHK is capableof upregulating a set of genes involved in tissue remodeling and theTGF-β signaling pathway which are downregulated during the progressionof emphysema. Accordingly, one embodiment provides a method for treatingCOPD or emphysema by administering a composition comprising thetripeptide GHK to a patient. In certain embodiments the compound furthercomprises a pharmaceutically acceptable carrier.

In certain embodiments, treating a patient having emphysema or COPD withGHK decreases an indicator, marker, symptom, or the severity of COPD oremphysema by at least 10%, e.g., by at least 20%, at least 30%, at least50%, at least 75%, at least 100%, at least 200% or more as compared tothe indicator, marker, symptom or the severity prior to treatment withGHK or as compared to patients not receiving treatment with GHK.

In certain embodiments, a composition comprising GHK is administered tothe airspace of the lung of a patient. In certain embodiments, acomposition comprising GHK is administered to the airspace of the lung.In certain embodiments, a composition comprising GHK is administeredorally or nasally. In certain embodiments, a composition comprising GHKis administered using an inhaler or a nebulizer. In certain embodiments,the GHK is not complexed with copper.

In certain embodiments, a composition comprises a therapeuticallyeffective dose of GHK. In certain embodiments, a composition comprisingGHK is administered to a patient at least once. In certain embodiments,a composition comprising GHK is administered to a patient repeatedly,e.g. once a day, twice a day, every other day, once a week, every 2weeks, once a month, etc.

In certain embodiments, GHK is used in the manufacture of a medicamentfor the treatment of emphysema or COPD. In certain embodiments, themedicament is administered to the airspace of the lung. In certainembodiments, the medicament is administered orally or nasally. Incertain embodiments, the medicament is administered using an inhaler ora nebulizer. In certain embodiments, the GHK is not complexed withcopper.

In certain embodiments, there is provided herein a method of enhancinglung tissue repair and/or healing by contacting the lung tissue of asubject with a GHK peptide. In certain embodiments the subject is amammal. In further embodiments, the subject is a human. In certainembodiments the subject has or has been diagnosed with or is at risk ofdeveloping emphysema. In certain embodiments the subject has or has beendiagnosed with or is at risk of developing COPD. In certain embodimentsthe method further comprises selecting a subject in need of reversal ofemphysematous lung destruction prior to administering to the subject acomposition comprising a GHK peptide. In certain embodiments,administering a composition comprising GHK to the subject reversesemphysematous lung destruction by at least 10%, e.g., by at least 20%,at least 30%, at least 50%, at least 75%, at least 100%, at least 200%or more as compared to lung tissue repair or healing prior toadministration of the composition or as compared to lung tissue repairor healing in patients not receiving treatment with the composition. Incertain embodiments, contacting the lung tissue of the patient with acomposition comprising GHK increases the lung tissue repair or healingby at least 10%, e.g., by at least 20%, at least 30%, at least 50%, atleast 75%, at least 100%, at least 200% or more as compared to lungtissue repair or healing prior to administration of the composition oras compared to lung tissue repair or healing in patients not receivingtreatment with the composition.

In certain embodiments, there is provided herein a method of enhancingthe repair of extracellular matrix in the lung tissue by contacting thelung tissue of a subject with a GHK peptide. In certain embodiments thesubject is a mammal. In further embodiments, the subject is a human. Incertain embodiments the subject has, has been diagnosed with, or is atrisk of developing emphysema. In certain embodiments the subject has,has been diagnosed with, or is at risk of developing COPD. In certainembodiments the method further comprises selecting a subject in need ofenhanced repair of extracellular matrix in the lung tissue prior tocontacting the lung tissue of the subject with the composition. Incertain embodiments, contacting the lung tissue of the patient with acomposition comprising GHK increases the repair of the extracellularmatrix by at least 10%, e.g., by at least 20%, at least 30%, at least50%, at least 75%, at least 100%, at least 200% or more as compared torepair of the extracellular matrix prior to administration of thecomposition or as compared to repair of the extracellular matrix inpatients not receiving treatment with the composition.

In certain embodiments, there is provided herein a method of increasingTGF-β signaling in the lung tissue by contacting the lung tissue of asubject with a GHK peptide. In certain embodiments the subject is amammal. In further embodiments, the subject is a human. In certainembodiments the subject has, has been diagnosed with, or is at risk ofdeveloping emphysema. In certain embodiments the subject has, has beendiagnosed with, or is at risk of developing COPD. In certain embodimentsthe method further comprises selecting a subject in need of increasedTGF-β signaling in the lung tissue prior to contacting the lung tissueof the subject with the composition. In certain embodiments, contactingthe lung tissue of the patient with a composition comprising GHKincreases TGF-β signaling by at least 10%, e.g., by at least 20%, atleast 30%, at least 50%, at least 75%, at least 100%, at least 200% ormore as compared to TGF-β signaling prior to administration of thecomposition or as compared to TGF-β signaling in patients not receivingtreatment with the composition.

In certain embodiments, there is provided herein an assay for assessingthe lungs of a subject comprising; 1) transforming the expressionproduct of at least two marker genes in a lung tissue sample obtainedfrom a subject into detectable targets wherein the marker genes areselected from Table 1 and/or Table 2, 2) measuring the level of thedetectable targets 3) comparing the level of the detectable targets inthe lung tissue sample from a subject to reference levels of thosedetectable targets, wherein a statistically significant difference inexpression levels of at least two detectable targets in the sample fromthe subject relative to the reference levels indicates the presence ofemphysema. In certain embodiments, one or more of the marker genes ofemphysematous damage are selected from the group consisting of ITGB1,NEDD9, ACVRL1, SMAD6 and TGFBR2.

In certain embodiments, the two or more marker genes of emphysematousdamage are selected from Table 1 and/or Table 2. In certain embodiments,one or more of the marker genes of emphysematous damage are selectedfrom the group consisting of ITGB1, NEDD9, ACVRL1, SMAD6 and TGFBR2. Incertain embodiments, one or more of these marker genes can be used inthe assays and systems described herein. In certain embodiments, one ormore of these marker genes and one or more additional genes can be usedin the assays and systems described herein.

In certain embodiments, the expression product of a marker gene is amRNA. In certain embodiments, the expression product of a marker gene isa protein.

In certain embodiments, the assay described above identifies patientshaving severe emphysema. In certain embodiments, the assay describedabove identifies a patient in need of a treatment for emphysema. Incertain embodiments, a subject indicated to have emphysema according tothe assay described above is administered a GHK tripeptide.

In certain embodiments, there is provided herein a computer implementedsystem for detecting emphysema in a subject, the system comprising; adetermination module configured to identify and detect the level ofexpression of at least two marker genes in a lung tissue sample obtainedfrom a subject wherein the marker genes are selected from Table 1 and/orTable 2, a storage module configured to store output data from thedetermination module, a comparison module adapted to identify from theoutput data whether the level of expression of at least two maker genesin the lung tissue sample obtained from a subject varies by astatistically significant amount from the expression level found in areference sample and a display module for displaying whether two or moremarker genes have a statistically significant variation in expressionlevel in the lung tissue sample obtained from a subject as compared tothe reference expression level and/or displaying the relative expressionlevels of the marker genes. In certain embodiments, if the computingmodule determines that the level of expression of at least two markergenes in the lung tissue sample obtained from a subject varies by astatistically significant amount as compared to the level of expressionthe in the reference sample, the display module displays a signalindicating the increased expression level in the sample obtained from asubject. In further embodiments, the signal indicates that the subjecthas an increased likelihood of having emphysema. In certain embodiments,one or more of the marker genes of emphysematous damage are selectedfrom the group consisting of ITGB1, NEDD9, ACVRL1, SMAD6 and TGFBR2.

In certain embodiments, the computer-implemented system displays asignal indicating that the expression levels in the sample obtained froma subject vary from those of the reference expression level. In certainembodiments, the computer-implemented system displays a signalindicating that the subject has an increased likelihood of havingemphysema. In certain embodiments, the computer-implemented systemdisplays a signal indicating that the subject is in need of treatmentfor emphysema. In certain embodiments, the computer-implemented systemdisplays a signal indicating the degree to which the expression levelsin the sample obtained from a subject vary from those of the referenceexpression level. In certain embodiments, the computer-implementedsystem displays a signal indicating that the subject has an increasedlikelihood of having a more severe case of emphysema.

The details of various embodiments of the invention are set forth in thedescription below. Other features, objects, and advantages of theinvention will be apparent from the description and the drawings, andfrom the claims.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is an illustrated overview of the study design.

FIGS. 2A-2C depict maps of gene expression relevance networks. Dark bluecircles indicate genes that have expression positively correlated withLm while circles with white centers indicate all other genes. Edges areindicated by lighter (positive correlation) or darker (negativecorrelation) lines.

FIGS. 3A-3E shows the relationship between gene expression changesassociated with regional emphysema severity (Lm) and cross-sectionalstudies of COPD-related gene expression using Gene Set EnrichmentAnalysis (GSEA). Black vertical lines represent the position of genesalong the ranked gene list. The lengths of the black lines correspond tothe magnitude of the running enrichment score from GSEA. Enrichmentswith an FDR q-value <0.05 were considered significant. FIG. 3A showsthat genes associated with Lm are enriched among the genes associatedwith emphysema or α-1 antitrypsin deficiency (Goplon et al., Americanjournal of respiratory cell and molecular biology 2004 31:595-600).T-statistic was calculated by a t-test between cases and controls. FIG.3B shows that genes associated with Lm are enriched among the genesassociated with diffusion capacity for carbon monoxide (DLCO) and forcedexpiratory volume at 1 sec (FEV1) (Spira et al American journal ofrespiratory cell and molecular biology 2004 31: 601-10), both of whichare pulmonary phenotypic expressions of COPD. T-statistics werecalculated by correlations of gene expression with a continuousvariable. FIG. 3C shows that genes associated with Lm are enriched amongthe genes associated with DLCO, FEV1, FEV1/FVC (FVC=forced vitalcapacity), GOLD2 level emphysema, and GOLD3 level emphysema (Wang, I.-M.et al. American journal of respiratory and critical care medicine 2008177:402-11). T-statistics for DLCO, FEV1 and FEV1/FVC were calculated byPearson correlation. T-statistics between COPD cases and never-smokedcontrols were calculated using a t-test. FIG. 3D shows that genesassociated with Lm are enriched among the genes associated withFEV1/FVC, FEV1, and COPD diagnosis (Bhattacharya et al., Americanjournal of respiratory cell and molecular biology 2009 40:359-67).T-statistics for FEV1 and FEV1/FVC were calculated by Pearsoncorrelation. T-statistics between cases and controls were calculatedusing a t-test. FIG. 3E shows that genes previously found to beassociated with COPD-related clinical variables are enriched among thegenes associated with Lm. T-statistic was calculated by correlations ofgene expression with Lm using mixed-effect linear models.

FIG. 4 shows correlations demonstrating the relationship between geneexpression changes associated with Lm and the cross-sectional stuffy ofCOPD-related gene expression from Goplon et al., (American journal ofrespiratory cell and molecular biology 2004 31:595-600). The gradatedbar represents the t-statistic from a t-test between five emphysemapatients and five non-smokers for 5,209 genes. The right end of the barindicates a more positive t-statistic and left indicates a more negativet-statistic (induced or repressed in COPD, respectively). The verticallines represent the position of genes associated with regional emphysemaseverity in the t-statistic ranking. The height of the vertical linescorresponds to the magnitude of the running enrichment score from GSEA.

FIG. 5 depicts correlations demonstrating the relationship between geneexpression changes associated with Lm and those induced by TGFβtreatment of A549 cells (Malizia et al., American journal of physiology.Lung cellular and molecular physiology 2008 295:L451-60). The gradatedbar represents the fold change between cell lines treated with andwithout TGFβ for 11910 genes. The vertical lines represent the positionof genes associated with regional emphysema severity in the t-statisticranking. The height of the vertical lines corresponds to the magnitudeof the running enrichment score from GSEA.

FIGS. 6A-6D depict the relationship between gene expression changesassociated with regional emphysema severity (Lm) and studies ofTGFβ-related gene expression using GSEA. The vertical lines representthe position of genes associated with regional emphysema severity in theranked gene list. The height of the vertical lines corresponds to themagnitude of the running enrichment score from GSEA. Enrichments with anFDR q-value <0.05 were considered significant. FIG. 6A shows that genesassociated with Lm are enriched among genes induced by TGFβ in Classenet al (Journal of immunology 2007 178:6931-40). FIG. 6B shows that genesassociated with Lm are enriched among genes induced by TGFβ in Koinumaet al (Molecular and cellular biology 2009 29:172-86). FIG. 6Cdemonstrates that genes associated with Lm are enriched among genesinduced by TGFβ in Malizia et al., (American journal of physiology. Lungcellular and molecular physiology 2008 295:L451-60). In FIGS. 6A-6C, thegradated bar represents the fold change between treated and untreatedsamples. FIG. 6D shows that genes most induced by TGFβ in seven studiesare enriched among the genes that are associated with Lm. T-statisticwas calculated by correlations of gene expression with Lm usingmixed-effect linear models.

FIGS. 7A-7D depict the relationship between gene expression changesassociated with regional emphysema severity (Lm) and the gene expressionchanges that occur when fibroblast cell lines are treated with GHK orTGFβ. The vertical lines represent the position of genes associated withregional emphysema severity in the ranked gene list. The height of thevertical lines corresponds to the magnitude of the running enrichmentscore from GSEA. Enrichments with an FDR q-value <0.05 were consideredsignificant. FIG. 7A shows that genes whose expression levels increasein response to treatment with GHK or TGFβ are enriched among genes thatdecrease with increasing emphysema severity. The gradated bar representsthe T-statistics from correlations of gene expression with Lm usingmixed-effect linear models. In FIGS. 7B-7D, the gradated bar representsthe fold changes between treated and untreated samples. FIG. 7B showsthat genes which are differentially expressed in response to GHK in theCMap or to TGFβ treatment are enriched among genes that aredifferentially expressed in response to 0.1 nM GHK in fibroblast celllines. FIG. 7C demonstrates that genes which are differentiallyexpressed with TGFβ treatment or that are down-regulated with increasingemphysema severity are enriched among genes that are differentiallyexpressed in response to 10 nM GHK in fibroblast cell lines. FIG. 7Ddemonstrates that genes which are differentially expressed in responseto GHK are enriched among genes that are differentially expressed inresponse to TGFβ treatment in fibroblast cell lines.

FIGS. 8A-8B demonstrate the effect of GHK treatment on gene expressionin human lung fibroblasts (HFL-1). FIG. 8A depicts the quantification ofβ1-integrin protein levels, demonstrating an increase after treatment ofHLF-1 cells with GHK at 0.1 or 10 nM (p<0.01), treatment with TGFβ at 10ng/mL (p<0.05) and treatment with GHK at 0.1 nM or 10 nM in combinationwith TGFβ (p<0.001) compared to vehicle (DMSO) treated controls. FIG. 8Bis an immunoblot of one of the three replicates of FIG. 8A.

FIG. 9 is a diagram of an embodiment of a system for performing a methodfor assessing the state of the lungs in a subject.

FIG. 10 is a diagram of an embodiment of a comparison module asdescribed herein.

FIG. 11 is a diagram of an embodiment of an operating system andapplications for a computing system as described herein.

DETAILED DESCRIPTION OF THE INVENTION

One aspect of the invention relates to a method of using a GHKtripeptide to treat emphysema or to treat COPD and/or to reverseemphysematous lung damage in a subject.

Another aspect of the invention relates to a method of detecting thepresence of emphysema in a subject or determining if a subject is inneed of treatment for emphysema by measuring the expression level of atleast two marker genes selected from the group consisting of ITGB1,NEDD9, ACVRL1, SMAD6, TGFBR2 and the genes listed in Table 1 and Table2, and comparing those expression levels to the expression levels foundin a reference sample. Another aspect of the invention relates to amethod of detecting the severity of emphysema in a subject ordetermining if a subject is in need of treatment for emphysema bymeasuring the expression level of at least two marker genes selectedfrom the group consisting of ITGB1, NEDD9, ACVRL1, SMAD6, TGFBR2 and thegenes listed in Table 1 and Table 2, and comparing those expressionlevels to the expression levels found in a reference sample. Alsoprovided herein is a computer system for performing the measurement andcomparison of these expression levels.

Definitions

For convenience, the meaning of certain terms and phrases used in thespecification, examples, and appended claims, are provided below. Ifthere is an apparent discrepancy between the usage of a term in the artand its definition provided herein, the definition provided within thespecification shall prevail.

Definitions of common terms in cell biology and molecular biology can befound in “The Merck Manual of Diagnosis and Therapy”, 18th Edition,published by Merck Research Laboratories, 2006 (ISBN 0-911910-18-2);Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Biology,published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); andRobert A. Meyers (ed.), Molecular Biology and Biotechnology: aComprehensive Desk Reference, published by VCH Publishers, Inc., 1995(ISBN 1-56081-569-8); The ELISA guidebook (Methods in molecular biology149) by Crowther J. R. (2000); Fundamentals of RIA and Other LigandAssays by Jeffrey Travis, 1979, Scientific Newsletters; Immunology byWerner Luttmann, published by Elsevier, 2006. Definitions of commonterms in molecular biology are also be found in Benjamin Lewin, GenesIX, published by Jones & Bartlett Publishing, 2007 (ISBN-13:9780763740634); Kendrew et al. (eds.), Molecular Biology andBiotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8) and Current Protocols inProtein Sciences 2009, Wiley Intersciences, Coligan et al., eds.

Unless otherwise stated, the present invention was performed usingstandard procedures, as described, for example in Methods in Enzymology,Volume 289: Solid-Phase Peptide Synthesis, J. N. Abelson, M. I. Simon,G. B. Fields (Editors), Academic Press; 1st edition (1997) (ISBN-13:978-0121821906); U.S. Pat. Nos. 4,965,343, and 5,849,954; Maniatis etal., Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., USA (1982); Sambrook et al.,Molecular Cloning: A Laboratory Manual (2 ed.), Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., USA (1989); Davis et al.,Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc.,New York, USA (1986); or Methods in Enzymology: Guide to MolecularCloning Techniques Vol. 152, S. L. Berger and A. R. Kimmerl Eds.,Academic Press Inc., San Diego, USA (1987); Current Protocols in ProteinScience (CPPS) (John E. Coligan, et. al., ed., John Wiley and Sons,Inc.), Current Protocols in Cell Biology (CPCB) (Juan S. Bonifacino et.al. ed., John Wiley and Sons, Inc.), and Culture of Animal Cells: AManual of Basic Technique by R. Ian Freshney, Publisher: Wiley-Liss; 5thedition (2005), Animal Cell Culture Methods (Methods in Cell Biology,Vol. 57, Jennie P. Mather and David Barnes editors, Academic Press, 1stedition, 1998) which are all incorporated by reference herein in theirentireties.

The terms “decrease”, “reduced”, “reduction”, “decrease” or “inhibit”are all used herein generally to mean a decrease by a statisticallysignificant amount. However, for avoidance of doubt, “reduced”,“reduction” or “decrease” or “inhibit” means a decrease by at least 10%as compared to a reference level, for example a decrease by at leastabout 20%, or at least about 30%, or at least about 40%, or at leastabout 50%, or at least about 60%, or at least about 70%, or at leastabout 80%, or at least about 90% or up to and including a 100% decrease(e.g. absent level or non-detectable level as compared to a referencesample), or any decrease between 10-100% as compared to a referencelevel. In the context of a disease marker or symptom is meant astatistically significant decrease in such level. The decrease can be,for example, at least 10%, at least 20%, at least 30%, at least 40% ormore, and is preferably down to a level accepted as within the range ofnormal for an individual without such disorder.

The terms “increased”, “increase” or “enhance” or “activate” are allused herein to generally mean an increase by a statically significantamount; for the avoidance of any doubt, the terms “increased”,“increase” or “enhance” or “activate” means an increase of at least 10%as compared to a reference level, for example an increase of at leastabout 20%, or at least about 30%, or at least about 40%, or at leastabout 50%, or at least about 60%, or at least about 70%, or at leastabout 80%, or at least about 90% or up to and including a 100% increaseor any increase between 10-100% as compared to a reference level, or atleast about a 2-fold, or at least about a 3-fold, or at least about a4-fold, or at least about a 5-fold or at least about a 10-fold increase,or any increase between 2-fold and 10-fold or greater as compared to areference level.

As used herein, the term “administer” refers to the placement of acomposition into a subject by a method or route which results in atleast partial localization of the composition at a desired site suchthat desired effect is produced. A compound or composition describedherein can be administered by any appropriate route known in the artincluding, but not limited to, oral or parenteral routes, includingintravenous, intramuscular, subcutaneous, transdermal, airway (aerosol),pulmonary, nasal, rectal, and topical (including buccal and sublingual)administration.

Exemplary modes of administration include, but are not limited to,injection, infusion, instillation, inhalation, or ingestion. “Injection”includes, without limitation, intravenous, intramuscular, intraarterial,intrathecal, intraventricular, intracapsular, intraorbital,intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous,subcuticular, intraarticular, sub capsular, subarachnoid, intraspinal,intracerebrospinal, and intrasternal injection and infusion. Inpreferred embodiments, the compositions are administered by intravenousinfusion or injection.

As used herein in the context of expression, the terms “treat,”“treatment,” and the like, refer to a decrease in severity, indicators,symptoms, markers of COPD or emphysema as described herein. In thecontext of the present invention insofar as it relates to any of theconditions recited herein, the terms “treat,” “treatment,” and the likemean to relieve, alleviate, ameliorate, inhibit, slow down, reverse, orstop the progression, aggravation, deterioration, progression,anticipated progression or severity of at least one symptom orcomplication associated with COPD or emphysema. In one embodiment, thesymptoms of COPD or emphysema are alleviated by at least 5%, at least10%, at least 20%, at least 30%, at least 40%, or at least 50%.

As used herein, the phrase “therapeutically effective amount”,“effective amount” or “effective dose” refers to an amount that providesa therapeutic benefit in the treatment, prevention, or management ofCOPD or emphysema, e.g. an amount that provides a statisticallysignificant decrease in at least one symptom of COPD or emphysema.Determination of a therapeutically effective amount is well within thecapability of those skilled in the art. Generally, a therapeuticallyeffective amount can vary with the subject's history, age, condition,sex, as well as the severity and type of the medical condition in thesubject, and administration of other pharmaceutically active agents.

As used herein, the term “pharmaceutical composition” refers to theactive agent in combination with a pharmaceutically acceptable carrierof chemicals and compounds commonly used in the pharmaceutical industry.The term “pharmaceutically acceptable carrier” excludes tissue culturemedium.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically acceptable carrier” as used herein means apharmaceutically acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial, involved in carrying or transporting the subject agents fromone organ, or portion of the body, to another organ, or portion of thebody. Each carrier must be “acceptable” in the sense of being compatiblewith the other ingredients of the formulation, for example the carrierdoes not decrease the impact of the agent on the treatment. In otherwords, a carrier is pharmaceutically inert.

As used herein, a “subject” means a human or animal. Usually the animalis a vertebrate such as a primate, rodent, domestic animal or gameanimal. Primates include chimpanzees, cynomologous monkeys, spidermonkeys, and macaques, e.g., Rhesus. Rodents include mice, rats,woodchucks, ferrets, rabbits and hamsters. Domestic and game animalsinclude cows, horses, pigs, deer, bison, buffalo, feline species, e.g.,domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g.,chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon.Patient or subject includes any subset of the foregoing, e.g., all ofthe above, but excluding one or more groups or species such as humans,primates or rodents. In certain embodiments, the subject is a mammal,e.g., a primate, e.g., a human. The terms, “patient”, “individual” and“subject” are used interchangeably herein.

Preferably, the subject is a mammal. The mammal can be a human,non-human primate, mouse, rat, dog, cat, horse, or cow, but are notlimited to these examples. Mammals other than humans can beadvantageously used, for example, as subjects that represent animalmodels of COPD or emphysema. In addition, the methods described hereincan be used to treat domesticated animals and/or pets. A subject can bemale or female. A subject can be one who has been previously diagnosedwith or identified as suffering from or having COPD or emphysema or oneor more complications related to COPD or emphysema, and optionally, butneed not have already undergone treatment for COPD or emphysema or theone or more complications related to COPD or emphysema. A subject canalso be one who is not suffering from COPD or emphysema. A subject canalso be one who has been diagnosed as having a strong likelihood ofdeveloping or or identified as suffering from COPD or emphysema or oneor more complications related to COPD or emphysema. It can include onewho shows improvements in known COPD or emphysema risk factors as aresult of receiving one or more treatments for COPD or emphysema or oneor more complications related to COPD or emphysema. Alternatively, asubject can also be one who has not been previously diagnosed as havingCOPD or emphysema or one or more complications related to COPD oremphysema. For example, a subject can be one who exhibits one or morerisk factors for COPD or emphysema or one or more complications relatedto COPD or emphysema, or a subject who does not exhibit COPD oremphysema risk factors, or a subject who is asymptomatic for COPD oremphysema or one or more complications related to COPD or emphysema. Asubject can also be one who is suffering from or at risk of developingCOPD or emphysema or one or more complications related to COPD oremphysema. A subject can also be one who has been diagnosed with oridentified as having one or more complications related to COPD oremphysema, or alternatively, a subject can be one who has not beenpreviously diagnosed with or identified as having one or morecomplications related to COPD.

As used herein, the term “emphysematous lung damage” or “emphysematouslung destruction” refers to the degradation of the lung parenchyma andalveoli characteristic of emphysema and COPD. This destruction decreasesor destroys the ability of the affected lung tissue to perform gasexchange. Emphysematous lung damage can be measured by mean linearintercept (Lm) as described herein.

The terms “respiratory disorder” and “respiratory disease” are usedinterchangeably herein and refer to any condition and/or disorderrelating to respiration and/or the respiratory system. The respiratorydisorder can be allergic or non-allergic. In some embodiments, therespiratory disorder is selected from the group consisting of asthma,atopic asthma, non-atopic asthma, emphysema, bronchitis, chronicobstructive pulmonary disease (COPD), sinusitis, allergic rhinitis. Insome embodiments, the respiratory disorder is characterized by increasedresponsiveness of the tracheas and bronchi to various stimuli, i.e.,allergens, resulting in a widespread narrowing of the airways.

The term “COPD” is generally applied to chronic respiratory diseaseprocesses characterized by the persistent obstruction of bronchial airflow. COPD patients can suffer from conditions such as bronchitis,cystic fibrosis, asthma or emphysema.

The term “asthma” as used herein is defined as a disease of the airwaysthat is characterized by increased responsiveness of thetracheobronchial tree to a multiplicity of stimuli.

The term “allergic respiratory disorder” or “hypersensitivity disease”refers to allergic diseases and/or disorders of the lungs or respiratorysystem. Allergic disorders are characterized by hypersensitivity to anallergen.

The term “allergen” as used herein refers to an innocuous antigen thatinduces an allergic or hypersensitive reaction.

The term “non-allergic” as used herein refers to a respiratory disorderthat is not a result from or caused by an allergen. Thus, thenon-allergic respiratory disorder is 55 caused by other mechanisms notrelating to hypersensitivity to air innocuous agent or allergen.

The term “computer” can refer to any non-human apparatus that is capableof accepting a structured input, processing the structured inputaccording to prescribed rules, and producing results of the processingas output. Examples of a computer include: a computer; a general purposecomputer; a supercomputer; a mainframe; a super mini-computer; amini-computer; a workstation; a micro-computer; a server; an interactivetelevision; a hybrid combination of a computer and an interactivetelevision; and application-specific hardware to emulate a computerand/or software. A computer can have a single processor or multipleprocessors, which can operate in parallel and/or not in parallel. Acomputer also refers to two or more computers connected together via anetwork for transmitting or receiving information between the computers.An example of such a computer includes a distributed computer system forprocessing information via computers linked by a network.

The term “computer-readable medium” may refer to any storage device usedfor storing data accessible by a computer, as well as any other meansfor providing access to data by a computer. Examples of astorage-device-type computer-readable medium include: a magnetic harddisk; a floppy disk; an optical disk, such as a CD-ROM and a DVD; amagnetic tape; a memory chip.

The term “software” is used interchangeably herein with “program” andrefers to prescribed rules to operate a computer. Examples of softwareinclude: software; code segments; instructions; computer programs; andprogrammed logic.

The term a “computer system” may refer to a system having a computer,where the computer comprises a computer-readable medium embodyingsoftware to operate the computer.

The term “statistically significant” or “significantly” refers to astandard definition of statistical significance and generally means atwo standard deviation (2SD) below normal, or lower, concentration ofthe marker. The term refers to statistical evidence that there is adifference. It is defined as the probability of making a decision toreject the null hypothesis when the null hypothesis is actually true.The decision is often made using the p-value.

The terms “t-statistic” and “t-stat” are used herein interchangeably. Asused herein it can a test of statistical significance which uses aformula from which a t value is derived. The value is then compared witha set of t-distribution tables to see whether the null hypothesis shouldbe rejected or not.

Other than in the operating examples, or where otherwise indicated, allnumbers expressing quantities of ingredients or reaction conditions usedherein should be understood as modified in all instances by the term“about.” The term “about” when used in connection with percentages canmean ±1%.

The singular terms “a,” “an,” and “the” include plural referents unlesscontext clearly indicates otherwise. Similarly, the word “or” isintended to include “and” unless the context clearly indicatesotherwise. Although methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of thisdisclosure, suitable methods and materials are described below. Theabbreviation, “e.g.” is derived from the Latin exempli gratia, and isused herein to indicate a non-limiting example. Thus, the abbreviation“e.g.” is synonymous with the term “for example.”

All patents and other publications identified are expressly incorporatedherein by reference for the purpose of describing and disclosing, forexample, the methodologies described in such publications that might beused in connection with the present invention. These publications areprovided solely for their disclosure prior to the filing date of thepresent application. Nothing in this regard should be construed as anadmission that the inventors are not entitled to antedate suchdisclosure by virtue of prior invention or for any other reason. Allstatements as to the date or representation as to the contents of thesedocuments is based on the information available to the applicants anddoes not constitute any admission as to the correctness of the dates orcontents of these documents.

Treatment of COPD and/or Emphysema

COPD

Certain aspects of the invention provided herein relate to methods oftreating COPD in a subject. COPD can be characterized as a destructionof both small airways and parenchyma resulting in a progressiveimpairment in pulmonary function. The disease may be divided into twosubgroups, namely chronic bronchitis and emphysema. Chronic bronchitisis characterized by mucus hypersecretion from the conducting airways,inflammation and eventual scarring of the bronchi (airway tubes). Manypersons with COPD have a component of both of these conditions.

The interaction between parenchymal disease and the vasculature is oftenclinically evident by the observation that patients with severe COPDhave mild or moderate pulmonary hypertension at rest.Histopathologically and microscopically, the pulmonary vasculature inCOPD is typically characterized by initial thickening with smooth muscledeposition as well as a loss of both alveolar septal structures andmicrovasculature. Furthermore, in COPD it has been observed that bothalveolar septal and endothelial cells undergo apoptosis.

The presenting symptoms for COPD are typically breathlessnessaccompanied by a decline in FEV1 (i.e., forced expiratory volume in 1second). COPD patients have difficulty breathing because they developsmaller, inflamed air passageways and have partially destroyed alveoli.Chronic bronchitis can also be diagnosed by asking the patient whetherthey have a “productive cough,” i.e. one that yields sputum. Thepatients' symptoms are cough and expectoration of sputum. Chronicbronchitis can lead to more frequent and severe respiratory infections,narrowing and plugging of the bronchi, difficult breathing anddisability.

COPD patients are traditionally treated with bronchodilators and/orsteroids and evaluated by spirometry for the presence of airflowobstruction and reversibility. If airflow obstruction is present andreversibility less than 15%, particularly in a smoker, then they areoften diagnosed as having COPD.

Emphysema

Certain aspects of the invention provided herein relate to methods oftreating emphysema in a subject and/or to assessing the severity ofemphysemas in a patient. Emphysema is a chronic lung disease whichaffects the alveoli and/or the ends of the smallest bronchi. Thecondition is characterized by destructive changes and enlargement of thealveoli (air sacs) within the lungs. The lung loses its elasticity andtherefore these areas of the lungs become enlarged. These enlarged areastrap stale air and do not effectively exchange it with fresh air. Thisresults in difficult breathing and may result in insufficient oxygenbeing delivered to the blood. The predominant symptom in patients withemphysema is shortness of breath.

GHK

Certain aspects of the invention provided herein relate to methods oftreating emphysema and/or COPD in a subject by administering to thesubject a GHK tripeptide. GHK is comprised of a Glycine-Histidine-Lysinetripeptide. GHK may be synthesized by methods familiar to those skilledin the art or purchased commercially (#CG51068 RayBiotech, Inc.Norcross, Ga.).

Variations and modifications to GHK peptide to provide means fortargeting. For example, GHK can be linked with a molecularcounter-ligand, for example but not limited to, molecules which targetthe lung epithelium, to make GHK tissue specific.

In one embodiment, GHK is linked to a carrier to enhance itsbioavailability. Such carriers are known in the art and include poly(alkyl) glycol such as poly ethylene glycol (PEG) or methoxypolyethyleneglycol (mPEG) which can increase the in vivo half life of proteins towhich they are conjugated. Methods of PEGylation of a peptide are wellknown by one of ordinary skill in the art, and are considerations of,for example, how large a PEG polymer to use. In some embodiments, apeptide can be fused to serum albumin to increase the serum half-life oftherapeutic polypeptides and peptides.

It will be appreciated that the GHK peptide useful in the methods andcomposition as disclosed herein can optionally contain amino acids otherthan the 20 amino acids commonly referred to as the 20 naturallyoccurring amino acids.

In some embodiments, any of the amino acids of the GHK peptide,including the terminal amino acids, can be modified either by naturalprocesses such as glycosylation and other post-translationalmodifications, or by chemical modification techniques which are wellknown in the art. Even the common modifications that occur naturally inpolypeptides are too numerous to list exhaustively here, but they arewell described in basic texts and in more detailed monographs, as wellas in a voluminous research literature, and they are well known to thoseof skill in the art. Among the known modifications which can be presentin polypeptides of the present invention are, to name an illustrativefew, acetylation, acylation, ADP-ribosylation, amidation, covalentattachment of flavin, covalent attachment of a heme moiety, covalentattachment of a polynucleotide or polynucleotide derivative, covalentattachment of a lipid or lipid derivative, covalent attachment ofphosphotidylinositol, cross-linking, cyclization, disulfide bondformation, demethylation, formation of covalent cross-links, formationof cysteine, formation of pyroglutamate, formylation,gamma-carboxylation, glycation, glycosylation, hydroxylation,iodination, methylation, myristoylation, oxidation, proteolyticprocessing, phosphorylation, prenylation, racemization, selenoylation,sulfation, transfer-RNA mediated addition of amino acids to proteinssuch as arginylation, and ubiquitination.

Such modifications are well known to those of skill and have beendescribed in great detail in the scientific literature. Severalparticularly common modifications, glycosylation, lipid attachment,sulfation, gamma-carboxylation of glutamic acid residues, hydroxylationand ADP-ribosylation, for instance, are described in most basic texts,such as, for instance, 1. E. Creighton, Proteins-Structure and MolecularProperties, 2nd Ed., W.H. Freeman and Company, New York, 1993. Manydetailed reviews are available on this subject, such as, for example,those provided by Wold, F., in Posttranslational Covalent Modificationof Proteins, B. C. Johnson, Ed., Academic Press, New York, pp 1 -12,1983; Sifter et al., Meth. Enzymol. 182: 626-646, 1990 and Rattan etal., Protein Synthesis: Posttranslational Modifications and Aging, Ann.N.Y. Acad. Sci. 663: 48-62, 1992.

It will also be appreciated, as is well known and as noted above, thatpeptides and polypeptides are not always entirely linear. For instance,polypeptides can be branched as a result of ubiquitination, and they canbe circular, with or without branching, generally as a result ofposttranslational events, including natural processing events and eventsbrought about by human manipulation which do not occur naturally.Circular, branched and branched circular polypeptides can be synthesizedby non translational natural processes and by entirely syntheticmethods.

Modifications can occur anywhere in a polypeptide, including the peptidebackbone, the amino acid side-chains and the amino or carboxyl termini.In fact, blockage of the amino or carboxyl group in a polypeptide, orboth, by a covalent modification, is common in naturally occurring and;synthetic polypeptides and such modifications can be present inpolypeptides of the present invention, as well. For instance, the aminoterminal residue of polypeptides made in E. coli, prior to proteolyticprocessing, almost invariably will be N-formylmethionine.

The modifications that occur in a polypeptide often will be a functionof how it is made. For polypeptides made by expressing a cloned gene ina host, for instance, the nature and extent of the modifications inlarge part will be determined by the host cell posttranslationalmodification capacity and the modification signals present in thepolypeptide amino acid sequence. For instance, as is well known,glycosylation often does not occur in bacterial hosts such as E. coli.Accordingly, when glycosylation is desired, a polypeptide should beexpressed in a glycosylation host, generally a eukaryotic cell. Insectcells often carry out the same posttranslational glycosylation asmammalian cells and, for this reason, insect cell expression systemshave been developed to efficiently express mammalian proteins havingnative patterns of glycosylation, inter alia. Similar considerationsapply to other modifications.

It will be appreciated that the same type of modification can be presentto the same or varying degree at several sites in a given polypeptide.Also, a given peptide or polypeptide can contain many types ofmodifications.

In some embodiments, N-methyl and hydroxy-amino acids can be substitutedfor conventional amino acids in solid phase peptide synthesis. However,production of polymers with reduced peptide bonds requires synthesis ofthe dimmer of amino acids containing the reduced peptide bond. Suchdimers are incorporated into polymers using standard solid phasesynthesis procedures. Other synthesis procedures are well known in theart.

Accordingly, functional derivatives of the GHK peptide may be preparedby modification of the amino acids of GHK peptide are encompassed foruse in the methods and compositions as disclosed herein. Modificationsmay occur anywhere in the GHK peptide sequence or its functionalderivative polypeptide, including the peptide backbone, the amino acidside-chains and the amino or carboxyl termini. Modifications mayinclude, for example, acetylation, acylation, ADP-ribosylation,amidation, covalent attachment of other functional moiety, covalentattachment of a lipid or lipid derivative, covalent attachment ofphosphotidylinositol, cross-linking, cyclization, disulfide bondformation, demethylation, formation of covalent cross-links,formylation, gamma-carboxylation, glycosylation,glycophosphatidylinositol (GPI) anchor formation, hydroxylation,iodination, methylation, myristoylation, oxidation, pegylation,proteolytic processing, phosphorylation, prenylation, racemization,selenoylation, sulfation, transfer-RNA mediated addition of amino acidsto proteins such as arginylation, and ubiquitination. See, for instance,E. Creighton Proteins-Structure and Molecular Properties, 2nd Ed., W. H.Freeman and Company, New York (1993); B. C. Johnson, Post TranslationalCovalent Modification of Proteins, Academic Press, New York, (1983);Seifter et al., Meth. Enzymol. 182: 626-646 (1990); Rattan et al., Ann.N. Y. Acad. Sci. 663: 48-62 (1992). Preparation of these modifiedderivatives may, for example, be useful if direct administration of theGHK peptide is contemplated.

In some embodiments the GHK peptide can be conjugated to a secondentity, for example, to promote stability or for specific cell typetargeting. In some embodiments, a GHK peptide or fragments, derivativesor variants thereof can be conjugated to a first fusion partner (i.e.IgG1 Fc). The conjugation can be a non-covalent or covalent interaction,for example, by means of chemical crosslinkage or conjugation. Asdiscussed herein, in some embodiments, the GHK peptide is fused to serumalbumin to increase the serum half-life of the GHK peptide.

In some embodiments, the GHK peptide can also be fused to a secondfusion partner, for example, to a polypeptide that targets the productto a desired location, or, for example, a tag that facilitates itspurification, if so desired. Tags and fusion partners can be designed tobe cleavable, if so desired. Another modification specificallycontemplated is attachment, e.g., covalent attachment, to a polymer. Inone aspect, polymers such as polyethylene glycol (PEG) ormethoxypolyethylene glycol (mPEG) can increase the in vivo half-life ofproteins to which they are conjugated. Methods of PEGylation ofpolypeptide agents are well known to those skilled in the art, as areconsiderations of, for example, how large a PEG polymer to use.

As used herein, the term “conjugate” or “conjugation” refers to theattachment of two or more entities to form one entity. For example, themethods of the present invention provide conjugation of a GHK peptide orfragments, derivatives or variants thereof joined with another entity,for example a moiety such as a first fusion partner that makes the GHKpeptide stable, such as Ig carrier particle, for example IgG1 Fc. Theattachment can be by means of linkers, chemical modification, peptidelinkers, chemical linkers, covalent or non-covalent bonds, or proteinfusion or by any means known to one skilled in the art. The joining canbe permanent or reversible. In some embodiments, several linkers can beincluded in order to take advantage of desired properties of each linkerand each protein in the conjugate. Flexible linkers and linkers thatincrease the solubility of the conjugates are contemplated for use aloneor with other linkers as disclosed herein. Peptide linkers can be linkedby expressing DNA encoding the linker to one or more proteins in theconjugate. Linkers can be acid cleavable, photocleavable and heatsensitive linkers. Methods for conjugation are well known by personsskilled in the art and are encompassed for use in the present invention.

According to the present invention, the GHK peptide or fragments,derivatives or variants thereof, can be linked to the first fusionpartner via any suitable means, as known in the art, see for exampleU.S. Pat. Nos. 4,625,014, 5,057,301 and 5,514,363, which areincorporated herein in their entirety by reference. For example, the GHKpeptide e can be covalently conjugated to the IgG1 Fc, either directlyor through one or more linkers. In one embodiment, a GHK peptide asdisclosed herein is conjugated directly to the first fusion partner(e.g. Fc), and in an alternative embodiment, a GHK peptide as disclosedherein can be conjugated to a first fusion partner (such as IgG1 Fc) viaa linker, e.g. a transport enhancing linker.

A large variety of methods for conjugation of a GHK peptide as disclosedherein with a first fusion partner (e.g. Fc) are known in the art. Suchmethods are e.g. described by Hermanson (1996, Bioconjugate Techniques,Academic Press), in U.S. Pat. No. 6,180,084 and U.S. Pat. No. 6,264,914which are incorporated herein in their entirety by reference and includee.g. methods used to link haptens to carriers proteins as routinely usedin applied immunology (see Harlow and Lane, 1988, “Antibodies: Alaboratory manual”, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.). It is recognized that, in some cases, a GHK peptide canlose efficacy or functionality upon conjugation depending, e.g., on theconjugation procedure or the chemical group utilized therein. However,given the large variety of methods for conjugation the skilled person isable to find a conjugation method that does not or least affects theefficacy or functionality of the entities, such as the GHK peptide to beconjugated.

Suitable methods for conjugation of a GHK peptide as disclosed hereinwith a first fusion partner (e.g. Fc) include e.g. carbodimideconjugation (Bauminger and Wilchek, 1980, Meth. Enzymol. 70: 151-159).Alternatively, a moiety can be coupled to a targeting agent as describedby Nagy et al., Proc. Natl. Acad. Sci. USA 93:7269-7273 (1996), and Nagyet al., Proc. Natl. Acad. Sci. USA 95:1794-1799 (1998), each of whichare incorporated herein by reference. Another method for conjugating onecan use is, for example sodium periodate oxidation followed by reductivealkylation of appropriate reactants and glutaraldehyde crosslinking.

One can use a variety of different linkers to conjugate a GHK peptide asdisclosed herein with a first fusion partner (e.g. Fc), for example butnot limited to aminocaproic horse radish peroxidase (HRP) or aheterobiofunctional cross-linker, e.g. carbonyl reactive andsulfhydryl-reactive cross-linker. Heterobiofunctional cross linkingreagents usually contain two reactive groups that can be coupled to twodifferent function targets on proteins and other macromolecules in a twoor three-step process, which can limit the degree of polymerizationoften associated with using homobiofunctional cross-linkers. Suchmulti-step protocols can offer a great control of conjugate size and themolar ratio of components.

The term “linker” refers to any means to join two or more entities, forexample a GHK peptide as disclosed herein with a first fusion partner(e.g. Fc). A linker can be a covalent linker or a non-covalent linker.Examples of covalent linkers include covalent bonds or a linker moietycovalently attached to one or more of the proteins to be linked. Thelinker can also be a non-covalent bond, e.g. an organometallic bondthrough a metal center such as platinum atom. For covalent linkages,various functionalities can be used, such as amide groups, includingcarbonic acid derivatives, ethers, esters, including organic andinorganic esters, amino, urethane, urea and the like. To provide forlinking, the effector molecule and/or the probe can be modified byoxidation, hydroxylation, substitution, reduction etc. to provide a sitefor coupling. It will be appreciated that modification which do notsignificantly decrease the function of the GHK peptide as disclosedherein or the first fusion partner (e.g. Fc) are preferred.

The dosage ranges for the administration of GHK peptide depend upon theform of the protein, and its potency, as described further herein, andare amounts large enough to produce the desired effect in which thesymptoms, markers, or signs of emphysema and/or COPD are reduced. Thedosage should not be so large as to cause adverse side effects.Generally, the dosage can vary with the age, condition, and sex of thepatient and can be determined by one of skill in the art. The dosage canalso be adjusted by the individual physician in the event of anycomplication. Typically, the dosage ranges from 0.001 mg/kg body weightto 0.5 mg/kg body weight. In one embodiment, the dose range is from 5μg/kg body weight to 30 μg/kg body weight. The doses can be given once aday, less than once a day or multiple times a day in order to achieve atherapeutically effective dose.

With respect to the therapeutic methods of the invention, it is notintended that the administration of the GHK peptide be limited to aparticular mode of administration, dosage, or frequency of dosing; thepresent invention contemplates all modes of administration, includingintramuscular, intravenous, inhalation, intraperitoneal, intravesicular,intraarticular, intralesional, subcutaneous, or any other routesufficient to provide a dose adequate to treat the respiratory disorder,e.g., COPD and/or emphysema. The therapeutic may be administered to thepatient in a single dose or in multiple doses. When multiple doses areadministered, the doses may be separated from one another by, forexample, one hour, three hours, six hours, eight hours, one day, twodays, one week, two weeks, or one month. For example, the therapeuticmay be administered for, e.g., 2, 3, 4, 5, 6, 7, 8, 10, 15, 20, or moreweeks. It is to be understood that, for any particular subject, specificdosage regimes should be adjusted over time according to the individualneed and the professional judgment of the person administering orsupervising the administration of the compositions. For example, thedosage of the therapeutic can be increased if the lower dose does notprovide sufficient therapeutic activity.

While the attending physician ultimately will decide the appropriateamount and dosage regimen, therapeutically effective amounts of the GHKpeptide can be provided at a dose of 0.0001, 0.01, 0.01 0.1, 1, 5, 10,25, 50, 100, 500, or 1,000 mg/kg. Effective doses may be extrapolatedfrom dose-response curves derived from in vitro or animal model testbioassays or systems.

Dosages for a particular patient or subject can be determined by one ofordinary skill in the art using conventional considerations, (e.g. bymeans of an appropriate, conventional pharmacological protocol). Aphysician may, for example, prescribe a relatively low dose at first,subsequently increasing the dose until an appropriate response isobtained. The dose administered to a patient is sufficient to effect abeneficial therapeutic response in the patient over time, or, e.g., toreduce symptoms, or other appropriate activity, depending on theapplication. The dose is determined by the efficacy of the particularformulation, and the activity, stability or serum half-life of the GHKpeptide as disclosed herein, or functional derivatives thereof, and thecondition of the patient, as well as the body weight or surface area ofthe patient to be treated. The size of the dose is also determined bythe existence, nature, and extent of any adverse side-effects thataccompany the administration of a particular vector, formulation, or thelike in a particular subject. Therapeutic compositions comprising a GHKpeptide or functional derivatives thereof are optionally tested in oneor more appropriate in vitro and/or in vivo animal models of disease,such as mice exposed to cigarette smoke (Shapiro Chest 2000117:2235-75), to confirm efficacy, tissue metabolism, and to estimatedosages, according to methods well known in the art. In particular,dosages can be initially determined by activity, stability or othersuitable measures of treatment vs. non-treatment (e.g., comparison oftreated vs. untreated cells or animal models), in a relevant assay.Formulations are administered at a rate determined by the LD50 of therelevant formulation, and/or observation of any side-effects of a GHKpeptide or functional derivatives thereof at various concentrations,e.g., as applied to the mass and overall health of the patient.Administration can be accomplished via single or divided doses.

In determining the effective amount of a GHK peptide or functionalderivatives thereof to be administered in the treatment or prophylaxisof disease the physician evaluates circulating plasma levels,formulation toxicities, and progression of the disease.

Subjects Amenable

Certain aspects of the invention described herein relate toadministering a GHK tripeptide to a patient having emphysema and/or COPDor diagnosed as having emphysema and/or COPD or at risk of havingemphysema and/or COPD, and/or is in need of reversal of emphysematousdamage. Certain aspects of the invention described herein relate toassessing the presence or severity of emphysema in a subject.

Subjects having emphysema and/or COPD can be identified by a physicianusing current methods of diagnosing emphysema and/or COPD. Symptoms ofCOPD and emphysema which characterize these conditions and aid indiagnosis are described above.

Subjects at risk of having or developing COPD and/or emphysema includesubjects who have smoked tobacco or been exposed to tobacco smoke.Cigarette smoke is considered to be a major risk factor in thedevelopment of COPD and its effects on the lung epithelium have beenwell characterized. Without wishing to be constrained by theory, it isbelieved that cigarette smoke induces necrosis and apoptosis of bothepithelial and endothelial cells which contributes to the pathogenesisof COPD.

Additional factors which increase the likelihood of a subject developingCOPD and/or emphysema include, but are not limited to, asbestos,environmental factors, predisposed genetic factor (e.g. AAT deficiency),exposure to tobacco products, exposure to chemicals, pollutants, andother factors that are known to increase the risk of COPD. For example,smokers are at a higher risk in developing COPD compared to non-smokers.In one particular embodiment, a subject at risk of developing COPDand/or emphysema refers to a subject who has been smoking at least 1/2to one pack of cigarettes for at least 1 year, typically at least 3years, more typically at least 5 years, still more typically at least 10years, and most typically at least 20 years. In certain embodiments, asubject at risk of having or developing COPD and/or emphysema is asubject who has been exposed to asbestos or a subject having a decreasedlevel of AAT in the blood.

In some embodiments, subjects at risk of having or developing COPDand/or emphysema can be identified by measuring the levels of geneexpression products of biomarkers known to be correlated with COPDand/or emphysema and comparing them to a reference level of those geneexpression products. In some embodiments, subjects at risk of having ordeveloping COPD and/or emphysema can be identified using the assays forbiomarkers described elsewhere herein.

In some embodiments, a pharmaceutical composition comprising a GHKtripeptide is used to treat a respiratory disorder, e.g. emphysema. Insome embodiments, the subject is selected for having a respiratorydisorder before being administered a composition comprising a GHKtripeptide. In some embodiments, a composition comprising a GHKtripeptide is used to treat COPD or emphysema. In some embodiments, asubject has been screened and identified to have COPD or emphysema priorto administration of the composition comprising a GHK tripeptide. Insome embodiments, the pharmaceutical composition comprising a GHKtripeptide comprises additional agents to treat COPD or otherrespiratory disorders.

Pharmaceutical Formulations

In some embodiments, a pharmaceutical composition comprises a GHKtripeptide, and optionally a pharmaceutically acceptable carrier. Incertain embodiments, the GHK tripeptide is not complexed with copper.

The compositions encompassed by the invention may further comprise atleast one pharmaceutically acceptable excipient. Excipients useful forpreparing the dosages forms from the composition according to theinvention and the instruments necessary to prepare them are described inU.S. Publication No. 2003/0206954 and 2004/0052843, which areincorporated herein in their entirety by reference.

For administration to a subject, a GHK tripeptide can be provided inpharmaceutically acceptable compositions. A pharmaceutically acceptablecomposition can comprise a therapeutically-effective amount of a GHKtripeptide formulated together with one or more pharmaceuticallyacceptable carriers (additives) and/or diluents.

Some examples of materials which can serve aspharmaceutically-acceptable carriers include: (1) sugars, such aslactose, glucose and sucrose; (2) starches, such as corn starch andpotato starch; (3) cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, methylcellulose, ethyl cellulose,microcrystalline cellulose and cellulose acetate; (4) powderedtragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such asmagnesium stearate, sodium lauryl sulfate and talc; (8) excipients, suchas cocoa butter and suppository waxes; (9) oils, such as peanut oil,cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12)esters, such as ethyl oleate and ethyl laurate; (13) agar; (14)buffering agents, such as magnesium hydroxide and aluminum hydroxide;(15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18)Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21)polyesters, polycarbonates and/or polyanhydrides; (22) bulking agents,such as polypeptides and amino acids (23) serum component, such as serumalbumin, HDL and LDL; (22) C₂-C₁₂ alcohols, such as ethanol; and (23)other non-toxic compatible substances employed in pharmaceuticalformulations. Wetting agents, coloring agents, release agents, coatingagents, sweetening agents, flavoring agents, perfuming agents,preservative and antioxidants can also be present in the formulation.The terms such as “excipient”, “carrier”, “pharmaceutically acceptablecarrier” or the like are used interchangeably herein. In someembodiments, the carrier inhibits the degradation of the GHK tripeptide.

As described in detail below, the pharmaceutical compositions of thepresent invention comprising a GHK tripeptide can be speciallyformulated for administration to a subject in solid, liquid or gel form,including those adapted for the following: (1) oral administration, forexample, drenches (aqueous or non-aqueous solutions or suspensions),lozenges, dragees, capsules, pills, tablets (e.g., those targeted forbuccal, sublingual, and systemic absorption), boluses, powders,granules, pastes for application to the tongue; (2) parenteraladministration, for example, by subcutaneous, intramuscular, intravenousor epidural injection as, for example, a sterile solution or suspension,or sustained-release formulation; (3) topical application, for example,as a cream, ointment, or a controlled-release patch or spray applied tothe skin; (4) intravaginally or intrarectally, for example, as apessary, cream or foam; (5) sublingually; (6) ocularly; (7)transdermally; (8) transmucosally; or (9) nasally. Additionally, a GHKtripeptide can be implanted into a patient or injected using a drugdelivery system. See, for example, Urquhart, et al., Ann. Rev.Pharmacol. Toxicol. 24: 199-236 (1984); Lewis, ed. “Controlled Releaseof Pesticides and Pharmaceuticals” (Plenum Press, New York, 1981); U.S.Pat. No. 3,773,919; and U.S. Pat. No. 35 3,270,960. Examples of dosageforms include, but are not limited to: tablets; caplets; capsules, suchas hard gelatin capsules and soft elastic gelatin capsules; cachets;troches; lozenges; dispersions; suppositories; ointments; cataplasms(poultices); pastes; powders; dressings; creams; plasters; solutions;patches; aerosols (e.g., nasal sprays or inhalers); gels; liquids suchas suspensions (e.g., aqueous or non-aqueous liquid suspensions,oil-in-water emulsions, or water-in-oil liquid emulsions), solutions,and elixirs; and sterile solids (e.g., crystalline or amorphous solids)that can be reconstituted to provide liquid dosage forms.

In methods of treatment described herein, the administration of a GHKtripeptide can be for either “prophylactic” or “therapeutic” purpose.When provided prophylactically, a GHK tripeptide can be administered toa subject in advance of any symptom, e.g. a respiratory disorder, e.g.asthma attack or a diagnosis of emphysema and/or COPD. The prophylacticadministration of a GHK tripeptide serves to prevent a respiratorydisorder, as disclosed herein. When provided therapeutically, a GHKtripeptide is provided at (or after) the onset of a symptom orindication of respiratory disorder. Thus, a GHK tripeptide can beprovided prior to the onset of respiratory disorder, e.g., onset of COPDand/or emphysema.

Toxicity and therapeutic efficacy can be determined by standardpharmaceutical procedures in cell cultures or experimental animals,e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50.Compositions that exhibit large therapeutic indices are preferred.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED50 with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized.

The therapeutically effective dose can be estimated initially from cellculture assays. A dose may be formulated in animal models to achieve acirculating plasma concentration range that includes the IC50 (i.e., theconcentration of the therapeutic which achieves a half-maximalinhibition of symptoms) as determined in cell culture. Levels in plasmamay be measured, for example, by high performance liquid chromatography.The effects of any particular dosage can be monitored by a suitablebioassay. The dosage may be determined by a physician and adjusted, asnecessary, to suit observed effects of the treatment.

In certain embodiments, the effective dose of a composition comprisingGHK is administered to a patient once. In certain embodiments, theeffective dose of a composition comprising GHK is administered to apatient repeatedly. Patients can be administered a therapeutic amount ofa composition comprising GHK, such as 0.5 mg/kg, 1.0 mg/kg, 2.0 mg/kg,2.5 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 40mg/kg or 50 mg/kg. A composition comprising GHK can be administered overa period of time, such as over a 5 minute, 10 minute, 15 minute, 20minute, or 25 minute period. The administration is repeated, forexample, on a regular basis, such as hourly for 3 hours, 6 hours, 12hours or longer or such as biweekly (i.e., every two weeks) for onemonth, two months, three months, four months or longer. After an initialtreatment regimen, the treatments can be administered on a less frequentbasis. For example, after administration biweekly for three months,administration can be repeated once per month, for six months or a yearor longer. Administration of a composition comprising GHK can reducelevels of a marker or symptom of emphysema and/or COPD, e.g., coughingor lung function impairment by at least 10%, at least 15%, at least 20%,at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80% or at least 90% or more.

The dosage of a GHK tripeptide can be determined by a physician andadjusted, as necessary, to suit observed effects of the treatment. Withrespect to duration and frequency of treatment, it is typical forskilled clinicians to monitor subjects in order to determine when thetreatment is providing therapeutic benefit, and to determine whether toincrease or decrease dosage, increase or decrease administrationfrequency, discontinue treatment, resume treatment or make otheralteration to treatment regimen. The dosing schedule can vary from oncea week to daily depending on a number of clinical factors, such as thesubject's sensitivity to the polypeptides. The desired dose can beadministered at one time or divided into subdoses, e.g., 2-4 subdosesand administered over a period of time, e.g., at appropriate intervalsthrough the day or other appropriate schedule. Such sub-doses can beadministered as unit dosage forms. In some embodiments, administrationis chronic, e.g., one or more doses daily over a period of weeks ormonths. Examples of dosing schedules are administration daily, twicedaily, three times daily or four or more times daily over a period of 1week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months,5 months, or 6 months or more.

Combination Therapies

As disclosed herein, a GHK tripeptide can be administrated to a subjectalone, or optionally in combination (e.g. simultaneously with,sequentially or separately) with one or more pharmaceutically activeagents, e.g. a second therapeutic agent known to be beneficial intreating emphysema and/or COPD. For example, exemplary pharmaceuticallyactive compound include, but are not limited to, those found inHarrison's principals of Internal Medicine, 13^(th) Edition, Eds. T. R.Harrison et al. McGraw-Hill N.Y., N.Y.; Physicians Desk Reference,50^(th) Edition, 1997, Oradell N.J., Medical Economics Co.;Pharmacological Basis of Therapeutics, 8^(th) Edition, Goodman andGilman, 1990; United States Pharmacopeia, The National Formulary, USPXII NF XVII, 1990; current edition of Goodman and Oilman's ThePharmacological Basis of Therapeutics; and current edition of The MerckIndex, the complete contents of all of which are incorporated herein byreference. By way of non-limiting example, such therapeutic agentsinclude bronchodialators (e.g. short and long acting (β-2 stimulants),orally administered bronchodilators, anti-cholinergic agents (e.g.ipratoprium bromide, theophylline compounds or a combination), inhaledanti-cholinergic agents, steroids (oral or topical), especiallycorticosteroids, mucolytic agents (e.g., ambroxol, ergosterin,carbocysteine, iodinated glycerol), antibiotics, antifungals,moisterization by nebulization, anti-tussives, respiratory stimulants(e.g., doxapram, almitrine bismesylate), α-1 antitrypsin administration,fromoterol, budesonide, and/or fromoterol/budesonide combinationtherapy.

In certain embodiments, the composition comprising a GHK tripeptide canfurther comprise, for example, an asthma agent. Exemplary agents knownto treat asthma include, but are not limited to, mast cell degranulationagents (i.e., Cromylyn sodium or Nedocromil sodium), leukotrieneinhibitors (i.e., Monteleukast sodium, Zafirlukast, or Pranlukasthydrate), corticosteroids (i.e., Beclomethasone, Budesonide,Ciclesonide, Hydrolysable glucocorticoid, Triamcinolone acetonide,Flunisolide, Mometasone furcate, or Fluticasone propionate), IgE bindinginhibitors (i.e., Omalizumab), Adenosine A2 agonists, Anti-CD23antibody, E-Selectin antagonists, P-Selectin antagonists, L-Selectinantagonists, interleukin inhibitors/monoclonal antibodies, pulmonarysurfactants, neurokinin antagonists, NF-Kappa-B inhibitors, PDE-4inhibitors (i.e., Cilomilast, or Roflumilast), Thromboxan A2 inhibitors(i.e., Rama-go troban, or Seratrodast), tryptase inhibitors, VIPagonists or antisense agents.

In some embodiments, a composition comprising a GHK tripeptide and apharmaceutically active agent can be administrated to the subject in thesame pharmaceutical composition or in different pharmaceuticalcompositions (at the same time or at different times). Whenadministrated at different times, a composition comprising a GHKtripeptide and the additional pharmaceutically active agent can beadministered within 5 minutes, 10 minutes, 20 minutes, 60 minutes, 2hours, 3 hours, 4, hours, 8 hours, 12 hours, 24 hours of administrationof the other. When a composition comprising a GHK tripeptide and thepharmaceutically active agent are administered in differentpharmaceutical compositions, routes of administration can be different.For example, a composition comprising a GHK tripeptide can beadministered by any appropriate route known in the art including, butnot limited to oral or parenteral routes, including intravenous,intramuscular, subcutaneous, transdermal, airway (aerosol), pulmonary,nasal, rectal, and topical (including buccal and sublingual)administration, and the pharmaceutically active agent is administered bya different route, e.g. a route commonly used in the art foradministration of the pharmaceutically active agent.

In some embodiments, a composition comprising a GHK tripeptide canprecede, can be co-current with and/or follow the pharmaceuticallyactive agent by intervals ranging from minutes to weeks. In embodimentswhere a composition comprising a GHK tripeptide and a pharmaceuticallyactive agent are applied separately to a cell, tissue or organism, onewould generally ensure that a significant period of time did not expirebetween the time of each delivery, such that the composition comprisinga GHK tripeptide and a pharmaceutically active agent would still be ableto exert an advantageously combined effect on the cell, tissue ororganism.

In some embodiments, the invention contemplates the use of a compositioncomprising a GHK tripeptide and the practice of the methods describedherein in conjunction with other therapies such as surgery, e.g.,enlarging a sinus passage, remove obstructing bone or nasal polyps,mucosal stripping, removal of sinuses, bullectomy, lung volume reductionsurgery, or lung transplantation.

Aerosol Formulations

A composition comprising a GHK tripeptide can be administered directlyto the airways of a subject in the form of an aerosol or bynebulization. For use as aerosols, a GHK tripeptide in solution orsuspension may be packaged in a pressurized aerosol container togetherwith suitable propellants, for example, hydrocarbon propellants likepropane, butane, or isobutane with conventional adjuvants. A GHKtripeptide can also be administered in a non-pressurized form such as ina nebulizer or atomizer.

The term “nebulization” is well known in the art to include reducingliquid to a fine spray. Preferably, by such nebulization small liquiddroplets of uniform size are produced from a larger body of liquid in acontrolled manner. Nebulization can be achieved by any suitable meanstherefore, including by using many nebulizers known and marketed today.For example, an AEROMIST pneumatic nebulizer available from InhalationPlastic, Inc. of Niles, Ill. When the active ingredients are adapted tobe administered, either together or individually, via nebulizer(s) theycan be in the form of a nebulized aqueous suspension or solution, withor without a suitable pH or tonicity adjustment, either as a unit doseor multidose device.

As is well known, any suitable gas can be used to apply pressure duringthe nebulization, with preferred gases to date being those which arechemically inert to a modulator of a GHK tripeptide. Exemplary gasesincluding, but are not limited to, nitrogen, argon or helium can be usedto high advantage.

In some embodiments, a GHK tripeptide can also be administered directlyto the airways in the form of a dry powder. For use as a dry powder, aGHK tripeptide can be administered by use of an inhaler. Exemplaryinhalers include metered dose inhalers and dry powdered inhalers.

A metered dose inhaler or “MDI” is a pressure resistant canister orcontainer filled with a product such as a pharmaceutical compositiondissolved in a liquefied propellant or micronized particles suspended ina liquefied propellant. The propellants which can be used includechlorofluorocarbons, hydrocarbons or hydrofluoroalkanes. Especiallypreferred propellants are P134a (tetrafluoroethane) and P227(heptafluoropropane) each of which may be used alone or in combination.They are optionally used in combination with one or more otherpropellants and/or one or more surfactants and/or one or more otherexcipients, for example ethanol, a lubricant, an anti-oxidant and/or astabilizing agent. The correct dosage of the composition is delivered tothe patient.

A dry powder inhaler (i.e. Turbuhaler (Astra AB)) is a system operablewith a source of pressurized air to produce dry powder particles of apharmaceutical composition that is compacted into a very small volume.

Dry powder aerosols for inhalation therapy are generally produced withmean diameters primarily in the range of <5 μm. As the diameter ofparticles exceeds 3 μm, there is increasingly less phagocytosis bymacrophages. However, increasing the particle size also has been foundto minimize the probability of particles (possessing standard massdensity) entering the airways and acini due to excessive deposition inthe oropharyngeal or nasal regions.

Suitable powder compositions include, by way of illustration, powderedpreparations of a GHK tripeptide thoroughly intermixed with lactose, orother inert powders acceptable for intrabronchial administration. Thepowder compositions can be administered via an aerosol dispenser orencased in a breakable capsule which may be inserted by the patient intoa device that punctures the capsule and blows the powder out in a steadystream suitable for inhalation. The compositions can includepropellants, surfactants, and co-solvents and may be filled intoconventional aerosol containers that are closed by a suitable meteringvalve.

Aerosols for the delivery to the respiratory tract are known in the art.See for example, Adjei, A. and Garren, J. Pharm. Res., 1: 565-569(1990); Zanen, P. and Lamm, J.-W. J. Int. J. Pharm., 114: 111-115(1995); Gonda, I. “Aerosols for delivery of therapeutic an diagnosticagents to the respiratory tract,” in Critical Reviews in TherapeuticDrug Carrier Systems, 6:273-313 (1990); Anderson et al., Am. Rev.Respir. Dis., 140: 1317-1324 (1989)) and have potential for the systemicdelivery of peptides and proteins as well (Patton and Platz, AdvancedDrug Delivery Reviews, 8:179-196 (1992)); Timsina et. al., Int. J.Pharm., 101: 1-13 (1995); and Tansey, I. P., Spray Technol. Market,4:26-29 (1994); French, D. L., Edwards, D. A. and Niven, R. W., AerosolSci., 27: 769-783 (1996); Visser, J., Powder Technology 58: 1-10(1989)); Rudt, S. and R. H. Muller, J. Controlled Release, 22: 263-272(1992); Tabata, Y, and Y. Ikada, Biomed. Mater. Res., 22: 837-858(1988); Wall, D. A., Drug Delivery, 2: 10 1-20 1995); Patton, J. andPlatz, R., Adv. Drug Del. Rev., 8: 179-196 (1992); Bryon, P., Adv. Drug.Del. Rev., 5: 107-132 (1990); Patton, J. S., et al., Controlled Release,28: 15 79-85 (1994); Damms, B. and Bains, W., Nature Biotechnology(1996); Niven, R. W., et al., Pharm. Res., 12(9); 1343-1349 (1995); andKobayashi, S., et al., Pharm. Res., 13(1): 80-83 (1996), contents of allof which are herein incorporated by reference in their entirety.

Oral Dosage Formulations

Pharmaceutical compositions comprising a GHK tripeptide can also beformulated to be suitable for oral administration, for example asdiscrete dosage forms, such as, but not limited to, tablets (includingwithout limitation scored or coated tablets), pills, caplets, capsules,chewable tablets, powder packets, cachets, troches, wafers, aerosolsprays, or liquids, such as but not limited to, syrups, elixirs,solutions or suspensions in an aqueous liquid, a non-aqueous liquid, anoil-in-water emulsion, or a water-in-oil emulsion. Such compositionscontain a predetermined amount of the pharmaceutically acceptable saltof the disclosed compounds, and may be prepared by methods of pharmacywell known to those skilled in the art. See generally, Remington'sPharmaceutical Sciences, 18th ed., Mack Publishing, Easton, Pa. (1990).

Typical oral dosage forms of the compositions of the disclosure areprepared by combining the pharmaceutically acceptable salt of disclosedcompounds in an intimate admixture with at least one excipient accordingto conventional pharmaceutical compounding techniques. Excipients cantake a wide variety of forms depending on the form of the compositiondesired for administration. For example, excipients suitable for use inoral liquid or aerosol dosage forms include, but are not limited to,water, glycols, oils, alcohols, flavoring agents, preservatives, andcoloring agents. Examples of excipients suitable for use in solid oraldosage forms (e.g., powders, tablets, capsules, and caplets) include,but are not limited to, starches, sugars, microcrystalline cellulose,kaolin, diluents, granulating agents, lubricants, binders, anddisintegrating agents. Due to their ease of administration, tablets andcapsules represent the most advantageous solid oral dosage unit forms,in which case solid pharmaceutical excipients are used. If desired,tablets can be coated by standard aqueous or nonaqueous techniques.These dosage forms can be prepared by any of the methods of pharmacy. Ingeneral, pharmaceutical compositions and dosage forms are prepared byuniformly and intimately admixing the active ingredient(s) with liquidcarriers, finely divided solid carriers, or both, and then shaping theproduct into the desired presentation if necessary.

For example, a tablet can be prepared by compression or molding.Compressed tablets can be prepared by compressing in a suitable machinethe active ingredient(s) in a free-flowing form, such as a powder orgranules, optionally mixed with one or more excipients. Molded tabletscan be made by molding in a suitable machine a mixture of the powderedcompound moistened with an inert liquid diluent. Examples of excipientsthat can be used in oral dosage forms of the disclosure include, but arenot limited to, binders, fillers, disintegrants, and lubricants. Binderssuitable for use in pharmaceutical compositions and dosage formsinclude, but are not limited to, corn starch, potato starch, or otherstarches, gelatin, natural and synthetic gums such as acacia, sodiumalginate, alginic acid, other alginates, powdered tragacanth, guar gum,cellulose and its derivatives (e.g., ethyl cellulose, cellulose acetate,carboxymethyl cellulose calcium, sodium carboxymethyl cellulose),polyvinyl pyrrolidone, methyl cellulose, pre-gelatinized starch,hydroxypropyl methyl cellulose, (e.g., Nos. 2208, 2906, 2910),microcrystalline cellulose, and mixtures thereof.

Suitable forms of microcrystalline cellulose include, but are notlimited to, the materials sold as AVICEL-PH-101 , AVICEL-PH-103 AVICELRC-581 , and AVICEL-PH-105 (available from FMC Corporation, AmericanViscose Division, Avicel Sales, Marcus Hook, Pa., U.S.A.), and mixturesthereof. An exemplary suitable binder is a mixture of microcrystallinecellulose and sodium carboxymethyl cellulose sold as AVICEL RC-581.Suitable anhydrous or low moisture excipients or additives includeAVICEL-PH-103™ and Starch 1500 LM.

Examples of fillers suitable for use in the pharmaceutical compositionsand dosage forms disclosed herein include, but are not limited to, talc,calcium carbonate (e.g., granules or powder), microcrystallinecellulose, powdered cellulose, dextrates, kaolin, mannitol, silicicacid, sorbitol, starch, pre-gelatinized starch, and mixtures thereof.The binder or filler in pharmaceutical compositions of the disclosure istypically present in from about 50 to about 99 weight percent of thepharmaceutical composition or dosage form.

Disintegrants are used in the compositions of the disclosure to providetablets that disintegrate when exposed to an aqueous environment.Tablets that contain too much disintegrant may swell, crack, ordisintegrate in storage, while those that contain too little may beinsufficient for disintegration to occur and may thus alter the rate andextent of release of the active ingredient(s) from the dosage form.Thus, a sufficient amount of disintegrant that is neither too little nortoo much to detrimentally alter the release of the active ingredient(s)should be used to form solid oral dosage forms of the disclosure. Theamount of disintegrant used varies based upon the type of formulationand mode of administration, and is readily discernible to those ofordinary skill in the art. Typical pharmaceutical compositions comprisefrom about 0.5 to about 15 weight percent of disintegrant, preferablyfrom about 1 to about 5 weight percent of disintegrant.

Disintegrants that can be used to form pharmaceutical compositions anddosage forms of the disclosure include, but are not limited to, agar,alginic acid, calcium carbonate, microcrystalline cellulose,croscarmellose sodium, crospovidone, polacrilin potassium, sodium starchglycolate, potato or tapioca starch, other starches, pre-gelatinizedstarch, clays, other algins, other celluloses, gums, and mixturesthereof.

Lubricants that can be used to form pharmaceutical compositions anddosage forms of the disclosure include, but are not limited to, calciumstearate, magnesium stearate, mineral oil, light mineral oil, glycerin,sorbitol, mannitol, polyethylene glycol, other glycols, stearic acid,sodium lauryl sulfate, talc, hydrogenated vegetable oil (e.g., peanutoil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil, andsoybean oil), zinc stearate, ethyl oleate, ethyl laureate, agar, andmixtures thereof. Additional lubricants include, for example, a syloidsilica gel (AEROSIL® 200, manufactured by W. R. Grace Co. of Baltimore,Md.), a coagulated aerosol of synthetic silica (marketed by Degussa Co.of Piano, Tex.), CAB-O-SIL® (a pyrogenic silicon dioxide product sold byCabot Co. of Boston, Mass.), and mixtures thereof. If used at all,lubricants are typically used in an amount of less than about 1 weightpercent of the pharmaceutical compositions or dosage forms into whichthey are incorporated.

This disclosure further encompasses lactose-free pharmaceuticalcompositions and dosage forms, wherein such compositions preferablycontain little, if any, lactose or other mono-or di-saccharides. As usedherein, the term “lactose-free” means that the amount of lactosepresent, if any, is insufficient to substantially increase thedegradation rate of an active ingredient.

Lactose-free compositions of the disclosure can comprise excipientswhich are well known in the art and are listed in the USP (XXI)/NF(XVI), which is incorporated herein by reference. In general,lactose-free compositions comprise a pharmaceutically acceptable salt ofan HIF inhibitor, a binder/filler, and a lubricant in pharmaceuticallycompatible and pharmaceutically acceptable amounts. Preferredlactose-free dosage forms comprise a pharmaceutically acceptable salt ofthe disclosed compounds, microcrystalline cellulose, pre-gelatinizedstarch, and magnesium stearate.

This disclosure further encompasses anhydrous pharmaceuticalcompositions and dosage forms comprising the disclosed compounds asactive ingredients, since water can facilitate the degradation of somecompounds. For example, the addition of water (e.g., 5%) is widelyaccepted in the pharmaceutical arts as a means of simulating long-termstorage in order to determine characteristics such as shelf life or thestability of formulations over time. See, e.g., Jens T. Carstensen, DrugStability: Principles & Practice, 379-80 (2nd ed., Marcel Dekker, NY,N.Y.: 1995). Water and heat accelerate the decomposition of somecompounds. Thus, the effect of water on a formulation can be of greatsignificance since moisture and/or humidity are commonly encounteredduring manufacture, handling, packaging, storage, shipment, and use offormulations.

Anhydrous pharmaceutical compositions and dosage forms of the disclosurecan be prepared using anhydrous or low moisture containing ingredientsand low moisture or low humidity conditions. Pharmaceutical compositionsand dosage forms that comprise lactose and at least one activeingredient that comprises a primary or secondary amine are preferablyanhydrous if substantial contact with moisture and/or humidity duringmanufacturing, packaging, and/or storage is expected.

An anhydrous pharmaceutical composition should be prepared and storedsuch that its anhydrous nature is maintained. Accordingly, anhydrouscompositions are preferably packaged using materials known to preventexposure to water such that they can be included in suitable formularykits. Examples of suitable packaging include, but are not limited to,hermetically sealed foils, plastics, unit dose containers (e.g., vials)with or without desiccants, blister packs, and strip packs.

For oral administration, the dosage should contain at least at least0.1% of a GHK tripeptide. The percentage of a GHK tripeptide in thesecompositions may, of course, be varied and may conveniently be betweenabout 2% to about 60% of the weight of the unit. The amount of a GHKtripeptide in such therapeutically useful compositions is such that asuitable dosage will be obtained.

Controlled Release Forms

In some embodiments, a GHK tripeptide can be administered by controlled-or delayed-release means. Controlled-release pharmaceutical productshave a common goal of improving drug therapy over that achieved by theirnon-controlled release counterparts. Ideally, the use of an optimallydesigned controlled-release preparation in medical treatment ischaracterized by a minimum of drug substance being employed to cure orcontrol the condition in a minimum amount of time. Advantages ofcontrolled-release formulations include: 1) extended activity of thedrug; 2) reduced dosage frequency; 3) increased patient compliance; 4)usage of less total drug; 5) reduction in local or systemic sideeffects; 6) minimization of drug accumulation; 7) reduction in bloodlevel fluctuations; 8) improvement in efficacy of treatment; 9)reduction of potentiation or loss of drug activity; and 10) improvementin speed of control of diseases or conditions. Kim, Cherng-ju,Controlled Release Dosage Form Design, 2 (Technomic Publishing,Lancaster, Pa.: 2000).

Conventional dosage forms generally provide rapid or immediate drugrelease from the formulation. Depending on the pharmacology andpharmacokinetics of the drug, use of conventional dosage forms can leadto wide fluctuations in the concentrations of the drug in a patient'sblood and other tissues. These fluctuations can impact a number ofparameters, such as dose frequency, onset of action, duration ofefficacy, maintenance of therapeutic blood levels, toxicity, sideeffects, and the like. Advantageously, controlled-release formulationscan be used to control a drug's onset of action, duration of action,plasma levels within the therapeutic window, and peak blood levels. Inparticular, controlled- or extended-release dosage forms or formulationscan be used to ensure that the maximum effectiveness of a drug isachieved while minimizing potential adverse effects and safety concerns,which can occur both from under-dosing a drug (i.e., going below theminimum therapeutic levels) as well as exceeding the toxicity level forthe drug.

Most controlled-release formulations are designed to initially releasean amount of drug (active ingredient) that promptly produces the desiredtherapeutic effect, and gradually and continually release other amountsof drug to maintain this level of therapeutic or prophylactic effectover an extended period of time. In order to maintain this constantlevel of drug in the body, the drug must be released from the dosageform at a rate that will replace the amount of drug being metabolizedand excreted from the body. Controlled-release of an active ingredientcan be stimulated by various conditions including, but not limited to,pH, ionic strength, osmotic pressure, temperature, enzymes, water, andother physiological conditions or compounds.

A variety of known controlled- or extended-release dosage forms,formulations, and devices can be adapted for use with the salts andcompositions of the disclosure. Examples include, but are not limitedto, those described in U.S. Pat. Nos. 3,845,770; 3,916,899; 3,536,809;3,598,123; 4,008,719; 5,674,533; 5,059,595; 5,591,767; 5,120,548;5,073,543; 5,639,476; 5,354,556; 5,733,566; and 6,365,185 B1; each ofwhich is incorporated herein by reference. These dosage forms can beused to provide slow or controlled-release of one or more activeingredients using, for example, hydroxypropylmethyl cellulose, otherpolymer matrices, gels, permeable membranes, osmotic systems (such asOROS® (Alza Corporation, Mountain View, Calif. USA)), multilayercoatings, microparticles, liposomes, or microspheres or a combinationthereof to provide the desired release profile in varying proportions.Additionally, ion exchange materials can be used to prepare immobilized,adsorbed salt forms of the disclosed compounds and thus effectcontrolled delivery of the drug. Examples of specific anion exchangersinclude, but are not limited to, Duolite® A568 and Duolite® AP143(Rohm&Haas, Spring House, Pa. USA).

One embodiment of the disclosure encompasses a unit dosage form thatincludes a pharmaceutically acceptable salt of the disclosed compounds(e.g., a sodium, potassium, or lithium salt), or a polymorph, solvate,hydrate, dehydrate, co-crystal, anhydrous, or amorphous form thereof,and one or more pharmaceutically acceptable excipients or diluents,wherein the pharmaceutical composition or dosage form is formulated forcontrolled-release. Specific dosage forms utilize an osmotic drugdelivery system.

A particular and well-known osmotic drug delivery system is referred toas OROS® (Alza Corporation, Mountain View, Calif. USA). This technologycan readily be adapted for the delivery of compounds and compositions ofthe disclosure. Various aspects of the technology are disclosed in U.S.Pat. Nos. 6,375,978 B1; 6,368,626 B1; 6,342,249 B1; 6,333,050 B2;6,287,295 B1; 6,283,953 B1; 6,270,787 B1; 6,245,357 B1; and 6,132,420;each of which is incorporated herein by reference. Specific adaptationsof OROS® that can be used to administer compounds and compositions ofthe disclosure include, but are not limited to, the OROS® Push-Pull™,Delayed Push-Pull™, Multi-Layer Push-Pull™, and Push-Stick™ Systems, allof which are well known. See, e.g. worldwide website alza.com.Additional OROS® systems that can be used for the controlled oraldelivery of compounds and compositions of the disclosure includeOROS®-CT and L-OROS®; see, Delivery Times, vol. 11 , issue II (AlzaCorporation).

Conventional OROS® oral dosage forms are made by compressing a drugpowder (e.g., a GHK tripeptide which is a salt) into a hard tablet,coating the tablet with cellulose derivatives to form a semi-permeablemembrane, and then drilling an orifice in the coating (e.g., with alaser). Kim, Cherng-ju, Controlled Release Dosage Form Design, 231-238(Technomic Publishing, Lancaster, Pa.: 2000). The advantage of suchdosage forms is that the delivery rate of the drug is not influenced byphysiological or experimental conditions. Even a drug with apH-dependent solubility can be delivered at a constant rate regardlessof the pH of the delivery medium. But because these advantages areprovided by a build-up of osmotic pressure within the dosage form afteradministration, conventional OROS® drug delivery systems cannot be usedto effectively delivery drugs with low water solubility.

In some embodiments, a specific dosage form of the GHK tripeptidecompositions of the disclosure includes: a wall defining a cavity, thewall having an exit orifice formed or formable therein and at least aportion of the wall being semipermeable; an expandable layer locatedwithin the cavity remote from the exit orifice and in fluidcommunication with the semipermeable portion of the wall; a dry orsubstantially dry state drug layer located within the cavity adjacentthe exit orifice and in direct or indirect contacting relationship withthe expandable layer; and a flow-promoting layer interposed between theinner surface of the wall and at least the external surface of the druglayer located within the cavity, wherein the drug layer includes a GHKtripeptide, or a polymorph, solvate, hydrate, dehydrate, co-crystal,anhydrous, or amorphous form thereof. See U.S. Pat. No. 6,368,626, theentirety of which is incorporated herein by reference.

In another embodiment, a specific dosage form of the GHK tripeptideincludes: a wall defining a cavity, the wall having an exit orificeformed or formable therein and at least a portion of the wall beingsemipermeable; an expandable layer located within the cavity remote fromthe exit orifice and in fluid communication with the semipermeableportion of the wall; a drug layer located within the cavity adjacent theexit orifice and in direct or indirect contacting relationship with theexpandable layer; the drug layer comprising a liquid, active agentformulation absorbed in porous particles, the porous particles beingadapted to resist compaction forces sufficient to form a compacted druglayer without significant exudation of the liquid, active agentformulation, the dosage form optionally having a placebo layer betweenthe exit orifice and the drug layer, wherein the active agentformulation comprises a GHK tripeptide, or a polymorph, solvate,hydrate, dehydrate, co-crystal, anhydrous, or amorphous form thereof.See U.S. Pat. No. 6,342,249, the entirety of which is incorporatedherein by reference.

In some embodiments, a GHK tripeptide is administered to a subject bysustained release or in pulses. Pulse therapy is not a form ofdiscontinuous administration of the same amount of a composition overtime, but comprises administration of the same dose of the compositionat a reduced frequency or administration of reduced doses. Sustainedrelease or pulse administration is particularly preferred when therespiratory disorder occurs continuously in the subject, for examplewhere the subject has continuous symptoms of a respiratory disorder.Each pulse dose can be reduced and the total amount of drug administeredover the course of treatment to the patient is minimized.

In some embodiments, individual pulses can be delivered to the patientcontinuously over a period of several hours, such as about 2, 4, 6, 8,10, 12, 14 or 16 hours, or several days, such as 2, 3, 4, 5, 6, or 7days, preferably from about 1 hour to about 24 hours and more preferablyfrom about 3 hours to about 9 hours.

In some embodiments, an interval between pulses or an interval of nodelivery is greater than 24 hours and preferably greater than 48 hours,and can be for even longer such as for 3, 4, 5, 6, 7, 8, 9 or 10 days,two, three or four weeks or even longer. As the results achieved may besurprising, the interval between pulses, when necessary, can bedetermined by one of ordinary skill in the art. Often, the intervalbetween pulses can be calculated by administering another dose of thecomposition when the composition or the active component of thecomposition is no longer detectable in the patient prior to delivery ofthe next pulse. Intervals can also be calculated from the in vivohalf-life of the composition. Intervals may be calculated as greaterthan the in vivo half-life, or 2, 3, 4, 5 and even 10 times greater thecomposition half-life.

In some embodiments, the number of pulses in a single therapeuticregimen may be as little as two, but is typically from about 5 to 10, 10to 20, 15 to 30 or more. In fact, patients can receive drugs for lifeaccording to the methods of this invention without the problems andinconveniences associated with current therapies. Compositions can beadministered by most any means, but are preferable delivered to thepatient as an injection (e.g. intravenous, subcutaneous, andintraarterial), infusion or instillation. Various methods and apparatusfor pulsing compositions by infusion or other forms of delivery to thepatient are disclosed in U.S. Pat. Nos. 4,747,825; 4,723,958; 4,948,592;4,965,251 and 5,403,590. Sustained release can also be accomplished bymeans of an osmotic pump. In some embodiments, a GHK tripeptide isadministered over a period of several days, such as 2, 3, 4, 5, 6 or 7days.

Parenteral Dosage Forms

In some embodiments, parenteral dosage forms of a modulator of a GHKtripeptide can also be administered to a subject with a respiratorydisorder by various routes, including, but not limited to, subcutaneous,intravenous (including bolus injection), intramuscular, andintraarterial. Since administration of parenteral dosage forms typicallybypasses the patient's natural defenses against contaminants, parenteraldosage forms are preferably sterile or capable of being sterilized priorto administration to a patient. Examples of parenteral dosage formsinclude, but are not limited to, solutions ready for injection, dryproducts ready to be dissolved or suspended in a pharmaceuticallyacceptable vehicle for injection, suspensions ready for injection, andemulsions. In addition, controlled-release parenteral dosage forms canbe prepared for administration of a patient, including, but not limitedto, administration DUROS®-type dosage forms, and dose-dumping.

Suitable vehicles that can be used to provide parenteral dosage forms ofthe GHK as disclosed within are well known to those skilled in the art.Examples include, without limitation: sterile water; water for injectionUSP; saline solution; glucose solution; aqueous vehicles such as but notlimited to, sodium chloride injection, Ringer's injection, dextroseInjection, dextrose and sodium chloride injection, and lactated Ringer'sinjection; water-miscible vehicles such as, but not limited to, ethylalcohol, polyethylene glycol, and propylene glycol; and non-aqueousvehicles such as, but not limited to, corn oil, cottonseed oil, peanutoil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.

Compounds that alter or modify the solubility of a pharmaceuticallyacceptable salt of a GHK tripeptide as disclosed herein can also beincorporated into the parenteral dosage forms of the disclosure,including conventional and controlled-release parenteral dosage forms.

Topical, Transdermal and Mucosal Dosage Forms

In some embodiments, the GHK peptide can be administered to a subjecttopically. In some embodiments, topical dosage forms of the GHK peptideinclude, but are not limited to, creams, lotions, ointments, gels,shampoos, sprays, aerosols, solutions, emulsions, and other forms knownto one of skill in the art. See, e.g., Remington's PharmaceuticalSciences, 18th ed., Mack Publishing, Easton, Pa. (1990); andIntroduction to Pharmaceutical Dosage Forms, 4th ed., Lea & Febiger,Philadelphia, Pa. (1985). For non-sprayable topical dosage forms,viscous to semi-solid or solid forms comprising a carrier or one or moreexcipients compatible with topical application and having a dynamicviscosity preferably greater than water are typically employed. Suitableformulations include, without limitation, solutions, suspensions,emulsions, creams, ointments, powders, liniments, salves, and the like,which are, if desired, sterilized or mixed with auxiliary agents (e.g.,preservatives, stabilizers, wetting agents, buffers, or salts) forinfluencing various properties, such as, for example, osmotic pressure.Other suitable topical dosage forms include sprayable aerosolpreparations wherein the active ingredient, preferably in combinationwith a solid or liquid inert carrier, is packaged in a mixture with apressurized volatile (e.g., a gaseous propellant, such as freon), or ina squeeze bottle. Moisturizers or humectants can also be added topharmaceutical compositions and dosage forms if desired. Examples ofsuch additional ingredients are well known in the art. See, e.g.,Remington's Pharmaceutical Sciences, 18.sup.th Ed., Mack Publishing,Easton, Pa. (1990).

Transdermal and mucosal dosage forms of the compositions comprising amodulator of a GHK tripeptide as disclosed herein include, but are notlimited to, ophthalmic solutions, patches, sprays, aerosols, creams,lotions, suppositories, ointments, gels, solutions, emulsions,suspensions, or other forms known to one of skill in the art. See, e.g.,Remington's Pharmaceutical Sciences, 18th Ed., Mack Publishing, Easton,Pa. (1990); and Introduction to Pharmaceutical Dosage Forms, 4th Ed.,Lea & Febiger, Philadelphia, Pa. (1985). Dosage forms suitable fortreating mucosal tissues within the oral cavity can be formulated asmouthwashes, as oral gels, or as buccal patches. Additional transdermaldosage forms include “reservoir type” or “matrix type” patches, whichcan be applied to the skin and worn for a specific period of time topermit the penetration of a desired amount of active ingredient.

Examples of transdermal dosage forms and methods of administration thatcan be used to administer the active ingredient(s) of the disclosureinclude, but are not limited to, those disclosed in U.S. Pat. Nos.4,624,665; 4,655,767; 4,687,481; 4,797,284; 4,810,499; 4,834,978;4,877,618; 4,880,633; 4,917,895; 4,927,687; 4,956,171; 5,035,894;5,091,186; 5,163,899; 5,232,702; 5,234,690; 5,273,755; 5,273,756;5,308,625; 5,356,632; 5,358,715; 5,372,579; 5,421,816; 5,466;465;5,494,680; 5,505,958; 5,554,381; 5,560,922; 5,585,111; 5,656,285;5,667,798; 5,698,217; 5,741,511; 5,747,783; 5,770,219; 5,814,599;5,817,332; 5,833,647; 5,879,322; and 5,906,830, each of which areincorporated herein by reference in their entirety.

Suitable excipients (e.g., carriers and diluents) and other materialsthat can be used to provide transdermal and mucosal dosage formsencompassed by this disclosure are well known to those skilled in thepharmaceutical arts, and depend on the particular tissue or organ towhich a given pharmaceutical composition or dosage form will be applied.With that fact in mind, typical excipients include, but are not limitedto water, acetone, ethanol, ethylene glycol, propylene glycol,butane-1,3-diol, isopropyl myristate, isopropyl palmitate, mineral oil,and mixtures thereof, to form dosage forms that are non-toxic andpharmaceutically acceptable.

Depending on the specific tissue to be treated, additional componentsmay be used prior to, in conjunction with, or subsequent to treatmentwith a GHK tripeptide. For example, penetration enhancers can be used toassist in delivering the active ingredients to or across the tissue.Suitable penetration enhancers include, but are not limited to: acetone;various alcohols such as ethanol, oleyl, an tetrahydrofuryl; alkylsulfoxides such as dimethyl sulfoxide; dimethyl acetamide; dimethylformamide; polyethylene glycol; pyrrolidones such aspolyvinylpyrrolidone; Kollidon grades (Povidone, Polyvidone); urea; andvarious water-soluble or insoluble sugar esters such as TWEEN 80(polysorbate 80) and SPAN 60 (sorbitan monostearate).

The pH of a pharmaceutical composition or dosage form, or of the tissueto which the pharmaceutical composition or dosage form is applied, mayalso be adjusted to improve delivery of the active ingredient(s).Similarly, the polarity of a solvent carrier, its ionic strength, ortonicity can be adjusted to improve delivery. Compounds such asstearates can also be added to pharmaceutical compositions or dosageforms to advantageously alter the hydrophilicity or lipophilicity of theactive ingredient(s) so as to improve delivery. In this regard,stearates can serve as a lipid vehicle for the formulation, as anemulsifying agent or surfactant, and as a delivery-enhancing orpenetration-enhancing agent. Different hydrates, dehydrates,co-crystals, solvates, polymorphs, anhydrous, or amorphous forms of thepharmaceutically acceptable salt of a GHK tripeptide can be used tofurther adjust the properties of the resulting composition.

Assessing Emphysema in a Subject

As described herein, the inventors have identified certain genes whichare upregulated or downregulated to a statistically significant degreein tissue which is subject to emphysematous tissue damage as compared toa reference sample. Herein, we are sometimes referring to these genes asmarker genes to indicate their relation to being a marker for emphysema.Accordingly, some embodiments of the invention are generally related toassays, methods and systems for assessing the emphysematous state of thelung(s) of a subject. In certain embodiments, the assays, methods andsystems relate to identifying a subject with emphysema or a need fortreatment for emphysema. Certain embodiments of the invention arerelated to assays, methods and systems for identifying the severity ofemphysema in a sample taken from the lung of a subject. In certainembodiments, the assays, methods and systems are directed todetermination of the expression level of a gene product (e.g. proteinand/or gene transcript such as mRNA) in a biological sample of asubject. In certain embodiments the assays, methods, and systems aredirected to determination of the expression level of a gene product ofat least two genes in a biological sample of a subject, i.e. at leasttwo genes, at least three genes, at least four genes, at least fivegenes, at least six genes, at least seven genes, at least eight genes,at least nine genes, at least 10 genes . . . at least 15 genes, . . . atleast 25 genes, . . . at least 30 genes, or more genes, or any number ofgenes selected from any in a combination of Table 1 and Table 2 asdescribed herein. Preferably, one looks at a group of genes where someincrease in expression and others decrease in expression. In someembodiments, the expression level of a gene product of the same numberof genes from each of Tables 1 and 2 is determined, e.g. two genes fromeach table. In some embodiments, the expression level of a gene productof different numbers of genes from each of Tables 1 and 2 is determined,e.g. two genes from Table 1 and 4 genes from Table 2 or 6 genes fromTable 1 and 3 genes from Table 2. In some embodiments, the marker genesare selected from at least the group consisting of ITGB1, NEDD9, ACVRL1,SMAD6 and TGFBR2. In some embodiments, the assays, methods, and systemsdescribed herein are directed to determination of the expression levelof a gene product of at least two genes in a biological sample of asubject, e.g. at least two genes, or at least three genes, or at leastfour genes, or all of the following genes; ITGB1, NEDD9, ACVRL1, SMAD6and TGFBR2.

The term “Lm” which is used in Table 1, Table 2, and elsewhere hereinrefers to a continuous measure of the severity of emphysematous lungdamage and is a measure of the size of the alveole as measured by thedistance between alveolar walls. Mean linear intercept (Lm) was measuredat 20 regularly spaced intervals of each of the microCT scans of lungsamples adjacent to samples used for gene expression using a previouslyvalidated grid of test lines projected onto the image and a custom macrolinked to specialized software (ImagePro Plus; MediaCybernetics, SilverSpring, Md., USA). A grid of test lines of a known length was appliedonto the image. The number of intercepts between these lines and tissuewas counted with Lm calculated as the total length of the test linesdivided by the number of cross-overs with tissue (equal to the number ofintercepts divided by 2). The pattern of the group selected should beone that shows a change from a group of similar subjects that do nothave COPD and/or emphysema. Preferably, the expression level overall forthe group shows a pattern change, i.e. some genes overexpress, someunderexpress. More preferably, the overally change is statisticallysignificant.

TABLE 1 Emphysema/COPD Marker Genes which are Downregulated in Tissuewith Emphysematous Damage Log2 Fold Change in Gene Expression betweenLeast Log2 Fold Diseased Change in Tertile of Gene Samples VariantExpression/ and Most Protein Variant Protein Unit Diseased NCBI SEQ SEQSEQ Increase in Tertile of Reference SEQ ID ID ID Gene Lm Samples No: IDNO NOs: NOs: NOs: ITGB1 −0.24 −0.37 NM_002211 1 128 253- 379-380 254PAPSS2 −0.50 −0.65 NM_004670 2 129 255 381 WFDC1 −0.41 −0.53 NM_021197 3130 N/A N/A NOSTRIN −0.77 −0.99 NM_001039724 4 131 256- 382-384 258NDEL1 −0.25 −0.32 NM_001025579 5 132 259 385 STARD13 −0.30 −0.39NM_178006 6 133 260- 386-390 264 NEDD9 −0.49 −0.63 NM_006403 7 134 265-391-392 266 ASRGL1 −0.40 −0.52 NM_001083926 8 135 267 393 EDNRB −0.57−0.74 NM_000115 9 136 268- 394-396 270 KLF13 −0.24 −0.31 NM_015995 10137 N/A N/A STOM −0.22 −0.28 NM_004099 11 138 271 397 GPR4 −0.41 −0.52NM_005282 12 139 N/A N/A TMEM2 −0.41 −0.53 NM_001135820 13 140 272 398SYN2 −0.31 −0.40 NM_133625 14 141 273 399 MT1JP −1.33 −1.71 NR_036677 15N/A N/A N/A RTN4 −0.23 −0.29 NM_020532 16 142 274- 400-403 277 PAG1−0.41 −0.52 NM_018440 17 143 N/A N/A COL4A2 −0.31 −0.40 NM_001846 18 144N/A N/A FCN3 −0.62 −0.80 NM_003665 19 145 278 404 ABTB2 −0.15 −0.20NM_145804 20 146 N/A N/A STX12 −0.34 −0.44 NM_177424 21 147 N/A N/AACVRL1 −0.51 −0.65 NM_000020 22 148 279 405 NES −0.30 −0.39 NM_006617 23149 N/A N/A PLXNA2 −0.35 −0.45 NM_025179 24 150 N/A N/A PODXL −0.47−0.60 NM_001018111 25 151 280 406 PECAM1 −0.36 −0.46 NM_000442 26 152N/A N/A TACC1 −0.27 −0.34 NM_001146216 27 153 281- 407-408 282 C13orf15−0.43 −0.56 NM_014059 28 154 N/A N/A S100A8 −0.96 −1.24 NM_002964 29 155N/A N/A ENG −0.33 −0.43 NM_001114753 30 156 283 409 TPST2 −0.17 −0.22NM_001008566 31 157 284 410 KRT7 −0.55 −0.71 NM_005556 32 158 N/A N/ASYCP2L −0.43 −0.56 NM_001040274 33 159 N/A N/A MYH9 −0.19 −0.25NM_002473 34 160 N/A N/A BTNL3 −0.31 −0.40 NM_197975 35 161 N/A N/AWNT2B −0.32 −0.41 NM_004185 36 162 285 411 PHLDB1 −0.31 −0.40NM_001144758 37 163 286- 412−413 287 CYP4Z1 −0.35 −0.45 NM_178134 38 164N/A N/A SMAD6 −0.67 −0.86 NM_005585 39 165 288 414 PRKCE −0.38 −0.49NM_005400 40 166 N/A N/A RCOR1 −0.22 −0.28 NM_015156 41 167 N/A N/ALUZP1 −0.22 −0.29 NM_001142546 42 168 289 415 HERC3 −0.22 −0.29NM_014606 43 169 N/A N/A KIAA1432 −0.20 −0.26 NM_020829 44 170 290-416-417 291 CUGBP2 −0.20 −0.26 NM_001025077 45 171 292- 418-420 294CRMP1 −0.20 −0.25 NM_001014809 46 172 295 421 S1PR1 −0.48 −0.62NM_001400 47 173 N/A N/A GABARAPL1 −0.32 −0.41 NM_031412 48 174 N/A N/AHPCAL1 −0.23 −0.30 NM_134421 49 175 296 422 CSPG4 −0.31 −0.41 NM_00189750 176 N/A N/A DBC1 −0.65 −0.83 NM_014618 51 177 N/A N/A RAI2 −0.26−0.34 NM_001172739 52 178 297- 423-425 299 KL −0.37 −0.48 NM_004795 53179 N/A N/A STXBP6 −0.41 −0.53 NM_014178 54 180 N/A N/A COL4A1 −0.33−0.43 NM_001845 55 181 N/A N/A TAL1 −0.27 −0.35 NM_003189 56 182 N/A N/AQKI −0.27 −0.35 NM_006775 57 183 300- 426-428 302 ARHGEF10 −0.34 −0.44NM_014629 58 184 N/A N/A CMTM8 −0.31 −0.39 NM_178868 59 185 N/A N/AFOXF1 −0.24 −0.31 NM_001451 60 186 N/A N/A TMBIM1 −0.18 −0.23 NM_00215261 187 N/A N/A MAP2 −0.43 −0.55 NM_001039538 62 188 303- 429-431 305SH3BP5 −0.32 −0.41 NM_001018009 63 189 306 432 ATOH8 −0.36 −0.46NM_032827 64 190 N/A N/A C1orf55 −0.22 −0.29 NM_152608 65 191 N/A N/ACTTNBP2NL −0.28 −0.36 NM_018704 66 192 N/A N/A LPHN2 −0.48 −0.62NM_012302 67 193 N/A N/A VIPR1 −0.65 −0.84 NM_001251882 68 194 307-433-436 310 PKN1 −0.29 −0.37 NM_213560 69 195 311 437 ECHDC3 −0.27 −0.35NM_024693 70 196 N/A N/A ADRB1 −0.46 −0.59 NM_000684 71 197 N/A N/AGPR133 −0.28 −0.35 NM_198827 72 198 N/A N/A KIAA1772 −0.12 −0.16NM_001142966 73 199 N/A N/A PTPN12 −0.26 −0.34 NM_001131009 74 200 312-438-439 313 ZNF358 −0.19 −0.25 NM_018083 75 201 N/A N/A LRRC8A −0.37−0.48 NM_001127244 76 202 314- 440-441 315 MAOA −0.44 −0.57 NM_000240 77203 N/A N/A EPAS1 −0.32 −0.41 NM_001430 78 204 N/A N/A TGFBR2 −0.15−0.20 NM_001024847 79 205 316 442

TABLE 2 Emphysema/COPD Marker Genes which are Upregulated in Tissue withEmphysematous Damage Log2 Fold Change in Gene Expression between LeastLog2 Fold Diseased Change in Tertile of Gene Samples Expression/ andMost Unit Diseased SEQ Protein Variant Variant Increase in Tertile ofNCBI ID SEQ ID SEQ ID Protein SEQ Gene Lm Samples Ref No: NO: NOs: NOs:ID NOs: BCL11A 0.29 0.37 NM_022893   80 206 317-318 443-444 CCR7 0.460.60 NM_001838   81 207 N/A N/A KIAA0125 0.61 0.79 NM_014792   OBSOLETEN/A N/A N/A CD79B 0.16 0.21 NM_001039933 82 208 319-320 445-446 AMPD10.45 0.58 NM_000036   83 209 321 447 CES3 0.19 0.25 NM_024922   84 210322-323 448-449 KLHDC6 0.25 0.32 NM_207335   85 211 N/A N/A ATP2C2 0.310.40 NM_014861   86 212 N/A N/A DHRS9 0.74 0.95 NM_001142270 87 213324-326 450-452 FCRLA 0.29 0.38 NM_001184866 88 214 327-332 453-458 CD220.34 0.44 NM_001771   89 215 333-335 459-461 RBP5 0.35 0.45 NM_031491  90 216 N/A N/A FAIM3 0.42 0.54 NM_005449   91 217 336-337 462-463 PRRX10.32 0.41 NM_006902   92 218 338 464 SLC45A3 0.31 0.40 NM_033102   93219 N/A N/A IRF4 0.38 0.48 NM_002460   94 220 339 465 PNMAL1 0.26 0.33NM_001103149 95 221 340 466 PRDM15 0.11 0.14 NM_001040424 96 222 341 467PDGFRL 0.31 0.40 NM_006207   97 223 N/A N/A ANKS1B 0.20 0.26 NM_152788  98 224 342-352 468-478 TMEM200A 0.25 0.33 NM_052913   99 225 N/A N/ATMEM9 0.17 0.22 NM_016456   100 226 N/A N/A C17orf76 0.21 0.27NM_001113567 101 227 353 479 KLHL6 0.41 0.53 NM_130446   102 228 N/A N/AFOLH1 0.17 0.21 NM_001193471 103 229 354-357 480-483 CALB2 0.21 0.28NM_001740   104 230 358 484 CD79A 0.60 0.78 NM_001783   105 231 359 485DMRT2 0.14 0.18 NM_001130865 106 232 360-361 486-487 CXCL13 0.90 1.17NM_006419   107 233 N/A N/A UGT8 0.18 0.24 NM_001128174 108 234 362 488SIGLEC6 0.30 0.38 NM_001245   109 235 363-367 489-493 MAN1C1 0.15 0.19NM_020379   110 236 N/A N/A OSBPL3 0.19 0.24 NM_015550   111 237 368-370494-496 MGC29506 0.48 0.61 NM_016459   112 238 N/A N/A UNQ6228 0.28 0.36XR_110905   113 N/A N/A N/A DERL3 0.30 0.39 NM_001135751 114 239 371-372497-498 NOPE 0.16 0.21 NM_020962   115 240 N/A N/A C5orf20 0.22 0.28NM_130848   116 241 N/A N/A LOC130576 0.21 0.27 NM_177964   117 242 N/AN/A ZFYVE19 0.12 0.16 NM_001077268 118 243 N/A N/A CR2 0.44 0.57NM_001006658 119 244 373 499 GPR110 0.74 0.96 NM_153840   120 245 374500 C8orf34 0.38 0.49 NM_052958   121 246 375 501 SLC4A8 0.37 0.47NM_001039960 122 247 376 502 FAM129C 0.17 0.22 NM_173544   123 248 377503 TMEM182 0.17 0.22 NM_144632   124 249 N/A N/A GPR110 0.69 0.88NM_153840   125 250 378 504 RALGPS2 0.42 0.54 NM_152663   126 251 N/AN/A transcript 0.20 0.25 Genbank 127 252 N/A N/A 3782069, Ref GenBankNo: BC012036 BC012036

The gene names listed in Tables 1 and 2 are common names. NCBI Gene IDnumbers for each of the genes listed in Tables 1 and 2 can be obtainedby searching the “Gene” Database of the NCBI (available on the WorldWide Web at http://www.ncbi.nlm.nih.gov/) using the common name as thequery and selecting the first returned Homo sapiens gene.

In certain embodiments, the subject may be exhibiting a sign or symptomof emphysema. In certain embodiments, the subject may be asymptomatic ornot exhibit a sign or symptom of emphysema, but can be at risk ofdeveloping emphysema to due exposure to tobacco smoke, pollutants,asbestos or other risk factors as described herein.

In some embodiments, the methods and assays described herein include (a)transforming the gene expression product into a detectable gene target;(b) measuring the amount of the detectable gene target; and (c)comparing the amount of the detectable gene target to an amount of areference, wherein if the amount of the detectable gene target isstatistically different from that of the amount of the reference level,the subject is identified as having emphysema or is in need of atreatment to reverse emphysematous tissue damage.

In some embodiments, the reference can be a level of expression of themarker gene product in a normal healthy subject with no symptoms orsigns of emphysema. For example, a normal healthy subject has normallung function, and/or is not diagnosed with emphysema and/or COPD,and/or has no history of smoking tobacco, and/or has not been exposed toasbestos or other environmental factors known to contribute to emphysema(e.g. smoke, dust, fires, etc.). In some embodiments, the reference canalso be a level of expression of the marker gene product in a controlsample, a pooled sample of control individuals or a numeric value orrange of values based on the same. In some embodiments, the referencecan also be a level of expression of the marker gene product in a tissuesample taken from undiseased lung tissue of the subject. In certainembodiments, wherein the progression of emphysematous tissue damage in asubject is to be monitored over time, the reference can also be a levelof expression of the marker gene product in a tissue sample taken fromlung tissue of the subject at an earlier date.

Marker Genes

In certain embodiments, the marker gene(s) are selected from the geneslisted in Table 1 and/or Table 2. In certain embodiments, one or moremarker genes are selected from the group consisting of ITGB1, NEDD9,ACVRL1, SMAD6 and TGFBR2. In tissue with emphysema or emphysematoustissue damage, the marker genes listed in Table 1 can be downregulatedand those in Table 2 can be upregulated, e.g. for marker genes listed inTable 1, if the measured marker gene expression in a subject is lower ascompared to a reference level of that marker gene's expression, then thesubject is identified as having emphysema. Likewise, for marker geneslisted in Table 2, if the measured marker gene expression in a subjectis higher by as compared to a reference level of that marker gene'sexpression, then the subject is identified as having emphysema.Preferably, once looks at a statistically significant change. However,even if a few genes in a group do not differ from normal, a subject canbe identified as having emphysema or at risk for developing emphysema ifthe overall change of the group shows a significant change, preferably astatistically significant change.

In certain embodiments marker genes in Table 2 are upregulated in tissuewith emphysematous damage. If the level of a gene expression product ofa marker gene in Table 2 is higher than a reference level of that markergene, the subject is more likely to have emphysema or to be in need of atreatment for emphysema. The level of a gene expression product of amarker gene in Table 2 which is higher than a reference level of thatmarker gene by at least about 10% than the reference amount, at leastabout 20%, at least about 30%, at least about 40%, at least about 50%,at least about 80%, at least about 100%, at least about 200%, at leastabout 300%, at least about 500% or at least about 1000% or more, isindicative that the subject has emphysema or emphysematous damage.

In certain embodiments marker genes in Table 1 are downregulated intissue with emphysematous damage. If the level of a gene expressionproduct of a marker gene in Table 1 is lower than a reference level ofthat marker gene, the subject is more likely to have emphysema or to bein need of a treatment for emphysema. The level of a gene expressionproduct of a marker gene in Table 1 which is lower than a referencelevel of that marker gene by at least about 10% than the referenceamount, at least about 20%, at least about 30%, at least about 40%, atleast about 50%, at least about 60%, at least about 70%, at least about80%, at least about 90%, or at least about 95%, about 98%, about 99% or100%, including all the percentages between 10-100% is indicative thatthe subject has emphysema or emphysematous damage.

In certain embodiments a subject is indicated to have emphysematousdamage, and/or to have emphysema, and/or to be in need of treatment foremphysematous damage, and/or to be in need of treatment for COPD and/oremphysema, and/or to have an increased likelihood of having emphysema,and/or to have an increased likelihood of having a more severe case ofemphysema if the expression level of one or more marker genes in asample obtained from a subject differs from the expression level in areference sample by a statistically significant amount.

In certain embodiments a sample is indicated to have emphysematousdamage, and/or to have emphysema, and/or to be in need of treatment foremphysematous damage, and/or to be in need of treatment for COPD and/oremphysema, and/or to have an increased likelihood of having emphysema,and/or to have an increased likelihood of having a more severe case ofemphysema if the log2 fold change in expression level of one or moremarker genes in a sample obtained from a patient as compared to theexpression level in a reference sample is at least the amount indicatedfor those particular marker genes in the column labeled “Log2 FoldChange in Gene Expression/Unit Increase in Lm” of Table 1 or Table 2.One looks at the overall changes in the group and if there is a changeof 2 orders of magnitude for the entire group, the individual isidentified as having a risk of emphysema, even if there is not astatistically significant change for a few of the individual genes.

In certain embodiments a subject is indicated to have emphysematousdamage, and/or to have emphysema, and/or to be in need of treatment foremphysematous damage, and/or to be in need of treatment for COPD and/oremphysema, and/or to have an increased likelihood of having emphysema,and/or to have an increased likelihood of having a more severe case ofemphysema if the log2 fold change in expression level of one or moremarker genes in a sample obtained from a patient as compared to theexpression level in a reference sample is at least 50% of the amountindicated for those particular marker genes in the column labeled “Log2Fold Change in Gene Expression/Unit Increase in Lm” of Table 1 or Table2. In certain embodiments a subject is indicated to have emphysematousdamage, and/or to have emphysema, and/or to be in need of treatment foremphysematous damage, and/or to be in need of treatment for COPD and/oremphysema, and/or to have an increased likelihood of having emphysema,and/or to have an increased likelihood of having a more severe case ofemphysema if the log2 fold change in expression level of one or moremarker genes in a sample obtained from a patient as compared to theexpression level in a reference sample is at least 60% of the amountindicated for those particular marker genes in the column labeled “Log2Fold Change in Gene Expression/Unit Increase in Lm” of Table 1 or Table2.

In certain embodiments a sample is indicated to have emphysematousdamage, and/or to have emphysema, and/or to be in need of treatment foremphysematous damage, and/or to be in need of treatment for COPD and/oremphysema, and/or to have an increased likelihood of having emphysema,and/or to have an increased likelihood of having a more severe case ofemphysema if the log2 fold change in expression level of one or moremarker genes in a sample obtained from a patient as compared to theexpression level in a reference sample is at least the amount indicatedfor those particular marker genes in the column labeled “Log2 FoldChange in Gene Expression between Least Diseased Tertile of Samples andMost Diseased Tertile of Samples” of Table 1 or Table 2.

Methods for Measuring Gene Expression Products Described Herein

As used herein, the term “transforming” or “transformation” refers tochanging an object or a substance, e.g., biological sample, nucleic acidor protein, into another substance. The transformation can be physical,biological or chemical. Exemplary physical transformation includes, butnot limited to, pre-treatment of a biological sample, e.g., from wholeblood to blood serum by differential centrifugation. Abiological/chemical transformation can involve at least one enzymeand/or a chemical reagent in a reaction. For example, a DNA sample canbe digested into fragments by one or more restriction enzyme, or anexogenous molecule can be attached to a fragmented DNA sample with aligase. In some embodiments, a DNA sample can undergo enzymaticreplication, e.g., by polymerase chain reaction (PCR).

Methods to measure gene expression products associated with the markergenes described herein are well known to a skilled artisan. Such methodsto measure gene expression products, e.g., protein level, include ELISA(enzyme linked immunosorbent assay), western blot, andimmunoprecipitation, immunofluorescence using detection reagents such asan antibody or protein binding agents. Alternatively, a peptide can bedetected in a subject by introducing into a subject a labeledanti-peptide antibody and other types of detection agent. For example,the antibody can be labeled with a radioactive marker whose presence andlocation in the subject is detected by standard imaging techniques.

For example, antibodies for ITGB1 (ab52971 AbCam Cambridge, Mass.) NEDD9(ab18056 AbCam Cambridge, Mass.), ACVRL1 (ab74039 AbCam Cambridge,Mass.), SMAD6 (ab13727 AbCam Cambridge, Mass.), and TGFBR2 (ab61213AbCam Cambridge, Mass.) are commercially available and can be used forthe purposes of the invention to measure protein expression levels.Alternatively, since the amino acid sequences for the marker genesdescribed herein are known and publically available at NCBI website, oneof skill in the art can raise their own antibodies against theseproteins of interest for the purpose of the invention.

The amino acid sequences of the marker genes described herein have beenassigned NCBI accession numbers for different species such as human,mouse and rat. In particular, the NCBI accession numbers for the aminoacid sequences of the human marker genes are included herein. For thehuman ITGB1 protein, the NCBI accession number for the amino acidsequence is NP_596867.1, NP_391988.1, or NP_002202.2 (due to thepresence of three transcript variants). For the human NEDD9 protein, theNCBI accession number for the amino acid sequence is eitherNP_001135865.1, NP_006394.1 or NP_892011.2 (due to the presence of threetranscript variants). For the human ACVRL1 protein, the NCBI accessionnumber for the amino acid sequence is either NP_000011.2 orNP_001070869.1 (due to the presence of two transcript variants). For thehuman SMAD6 protein, the NCBI accession number for the amino acidsequence is NP_001136333.1 and NP_005576.3 (due to the presence of twotranscript variants). For the human TGFBR2 protein, the NCBI accessionnumber for the amino acid sequence is NP_001020018.1 or NP_003233.4 (dueto the presence of two transcript variants).

In alternative embodiments, antibodies directed against peptides encodedby the gene of interest, for example an antibody against ACVRL1 (ab74039AbCam Cambridge, Mass.) can also be used in the assays and methodsdescribed herein. Such diagnostic methods can be used to detectabnormalities in the level of expression of the peptide, orabnormalities in the structure and/or tissue, cellular, or subcellularlocation of the peptide.

In another embodiment, immunohistochemistry (“IHC”) andimmunocytochemistry (“ICC”) techniques can be used. IHC is theapplication of immunochemistry to tissue sections, whereas ICC is theapplication of immunochemistry to cells or tissue imprints after theyhave undergone specific cytological preparations such as, for example,liquid-based preparations. Immunochemistry is a family of techniquesbased on the use of an antibody, wherein the antibodies are used tospecifically target molecules inside or on the surface of cells. Theantibody typically contains a marker that will undergo a biochemicalreaction, and thereby experience a change color, upon encountering thetargeted molecules. In some instances, signal amplification can beintegrated into the particular protocol, wherein a secondary antibody,that includes the marker stain or marker signal, follows the applicationof a primary specific antibody.

In certain embodiments, the gene expression products as described hereincan be instead determined by determining the level of messenger RNA(mRNA) expression of genes associated with the marker genes describedherein. Such molecules can be isolated, derived, or amplified from abiological sample, such as a lung biopsy. Detection of mRNA expressionis known by persons skilled in the art, and comprise, for example butnot limited to, PCR procedures, RT-PCR, Northern blot analysis,differential gene expression, RNA protection assay, microarray analysis,hybridization methods etc.

The nucleic acid sequences of the marker genes described herein havebeen assigned NCBI accession numbers for different species such ashuman, mouse and rat. In particular, the NCBI accession numbers for thenuclei acid sequences of the human marker genes are included herein. Forthe human ITGB1 mRNA, the NCBI accession number for the nucleic acidsequence is NM_002211.3, NM_033668.2, or NM_133376.2 each of whichrepresents a different transcript variant. For the human NEDD9 mRNA, theNCBI accession number for the nucleic acid sequence is eitherNM_182966.3, NM_006403.3, or NM_001142393.1, each of which represents adifferent transcript variant. For the human ACVRL1 mRNA, the NCBIaccession number for the nucleic acid sequence is NM_001077401.1 orNM_000020.2, each of which represents a different transcript variant.For the human SMAD6 mRNA, the NCBI accession number for the nucleic acidsequence is NM_005585.4 or NM_001142861.2, each of which represents adifferent transcript variant. For the human TGFBR2 mRNA, the NCBIaccession number for the nucleic acid sequence is NM_003242.5 orNM_001024847.2, each of which represents a different transcript variant.Accordingly, a skilled artisan can design an appropriate primer based onthe known sequence for determining the mRNA level of the respectivegene.

Nucleic acid and ribonucleic acid (RNA) molecules can be isolated from aparticular biological sample using any of a number of procedures, whichare well-known in the art, the particular isolation procedure chosenbeing appropriate for the particular biological sample. For example,freeze-thaw and alkaline lysis procedures can be useful for obtainingnucleic acid molecules from solid materials; heat and alkaline lysisprocedures can be useful for obtaining nucleic acid molecules fromurine; and proteinase K extraction can be used to obtain nucleic acidfrom blood (Roiff, A et al. PCR: Clinical Diagnostics and Research,Springer (1994)).

In general, the PCR procedure describes a method of gene amplificationwhich is comprised of (i) sequence-specific hybridization of primers tospecific genes within a nucleic acid sample or library, (ii) subsequentamplification involving multiple rounds of annealing, elongation, anddenaturation using a DNA polymerase, and (iii) screening the PCRproducts for a band of the correct size. The primers used areoligonucleotides of sufficient length and appropriate sequence toprovide initiation of polymerization, i.e. each primer is specificallydesigned to be complementary to each strand of the genomic locus to beamplified.

In an alternative embodiment, mRNA level of gene expression productsdescribed herein can be determined by reverse-transcription (RT) PCR andby quantitative RT-PCR (QRT-PCR) or real-time PCR methods. Methods ofRT-PCR and QRT-PCR are well known in the art.

Systems for Identifying a Subject with Increased Risk for HavingEmphysema or Needing Treatment for Emphysema

In another embodiment of the assays described herein, the assaycomprises or consists essentially of a system for transforming andmeasuring the amount of gene expression products of at least two markergenes as described herein and comparing them to a reference expressionlevel. If the comparison system, which can be a computer implementedsystem, indicates that the amount of the measured gene expressionproduct is statistically different from that of the reference amount,the subject from which the sample is collected can be identified ashaving an increased risk for having emphysema or for being in need of atreatment for emphysema.

Embodiments of the invention also provide for systems (and computerreadable media for causing computer systems) to perform a method forassessing the state of the lungs of a subject by measuring the level ofgene expression products of at least two marker genes selected fromTable 1 and/or Table 2. In certain embodiments, one or more of themarker genes are selected from the group consisting of ITGB1, NEDD9,ACVRL1, SMAD6 and TGFBR2.

In one embodiment, provided herein is a system comprising: (a) at leastone memory containing at least one computer program adapted to controlthe operation of the computer system to implement a method that includes(i) a determination module configured to identify and detect at theexpression level of at least two marker genes in a lung tissue sampleobtained from a subject; (ii) a storage module configured to storeoutput data from the determination module; (iii) a computing moduleadapted to identify from the output data whether the level of expressionof at least two marker genes in the lung tissue sample obtained from asubject varies by a statistically significant amount from the expressionlevel found in a reference sample and (iv) a display module fordisplaying whether two or more marker genes have a statisticallysignificant variation in expression level in the lung tissue sampleobtained from a subject as compared to the reference expression leveland/or displaying the relative expression levels of the marker genes and(b) at least one processor for executing the computer program (see FIG.9).

Embodiments of the invention can be described through functionalmodules, which are defined by computer executable instructions recordedon computer readable media and which cause a computer to perform methodsteps when executed. The modules are segregated by function for the sakeof clarity. However, it should be understood that the modules/systemsneed not correspond to discreet blocks of code and the describedfunctions can be carried out by the execution of various code portionsstored on various media and executed at various times. Furthermore, itshould be appreciated that the modules can perform other functions, thusthe modules are not limited to having any particular functions or set offunctions.

The computer readable storage media can be any available tangible mediathat can be accessed by a computer. Computer readable storage mediaincludes volatile and nonvolatile, removable and non-removable tangiblemedia implemented in any method or technology for storage of informationsuch as computer readable instructions, data structures, program modulesor other data. Computer readable storage media includes, but is notlimited to, RAM (random access memory), ROM (read only memory), EPROM(erasable programmable read only memory), EEPROM (electrically erasableprogrammable read only memory), flash memory or other memory technology,CD-ROM (compact disc read only memory), DVDs (digital versatile disks)or other optical storage media, magnetic cassettes, magnetic tape,magnetic disk storage or other magnetic storage media, other types ofvolatile and non-volatile memory, and any other tangible medium whichcan be used to store the desired information and which can accessed by acomputer including and any suitable combination of the foregoing.

Computer-readable data embodied on one or more computer-readable mediamay define instructions, for example, as part of one or more programsthat, as a result of being executed by a computer, instruct the computerto perform one or more of the functions described herein, and/or variousembodiments, variations and combinations thereof. Such instructions maybe written in any of a plurality of programming languages, for example,Java, J#, Visual Basic, C, C#, C++, Fortran, Pascal, Eiffel, Basic,COBOL assembly language, and the like, or any of a variety ofcombinations thereof. The computer-readable media on which suchinstructions are embodied may reside on one or more of the components ofeither of a system, or a computer readable storage medium describedherein, may be distributed across one or more of such components.

The computer-readable media may be transportable such that theinstructions stored thereon can be loaded onto any computer resource toimplement the aspects of the present invention discussed herein. Inaddition, it should be appreciated that the instructions stored on thecomputer-readable medium, described above, are not limited toinstructions embodied as part of an application program running on ahost computer. Rather, the instructions may be embodied as any type ofcomputer code (e.g., software or microcode) that can be employed toprogram a computer to implement aspects of the present invention. Thecomputer executable instructions may be written in a suitable computerlanguage or combination of several languages. Basic computationalbiology methods are known to those of ordinary skill in the art and aredescribed in, for example, Setubal and Meidanis et al., Introduction toComputational Biology Methods (PWS Publishing Company, Boston, 1997);Salzberg, Searles, Kasif, (Ed.), Computational Methods in MolecularBiology, (Elsevier, Amsterdam, 1998); Rashidi and Buehler,Bioinformatics Basics: Application in Biological Science and Medicine(CRC Press, London, 2000) and Ouelette and Bzevanis Bioinformatics: APractical Guide for Analysis of Gene and Proteins (Wiley & Sons, Inc.,2nd ed., 2001).

The functional modules of certain embodiments of the invention includeat minimum a determination module, a storage module, a computing module,and a display module. The functional modules can be executed on one, ormultiple, computers, or by using one, or multiple, computer networks.The determination module has computer executable instructions to providee.g., levels of expression products etc in computer readable form.

The determination module can comprise any system for detecting a signalelicited from the marker genes described herein in a biological sample.In some embodiments, such systems can include an instrument, e.g.,StepOnePlus Real-Time PCR systems (Applied Biosystems) as describedherein for quantitative RT-PCR. In another embodiment, the determinationmodule can comprise multiple units for different functions, such asamplification and hybridization. In one embodiment, the determinationmodule can be configured to perform the quantitative RT-PCR methodsdescribed in the Examples, including amplification, detection, andanalysis. In some embodiments, such systems can include an instrument,e.g., the Cell Biosciences NanoPro 1000 System (Cell Biosciences) forquantitative measurement of peptides and/or proteins.

In some embodiments, the determination module can be further configuredto identify and detect the presence of at least one additional emphysemarelated marker gene.

The information determined in the determination system can be read bythe storage module. As used herein the “storage module” is intended toinclude any suitable computing or processing apparatus or other deviceconfigured or adapted for storing data or information. Examples ofelectronic apparatus suitable for use with the present invention includestand-alone computing apparatus, data telecommunications networks,including local area networks (LAN), wide area networks (WAN), Internet,Intranet, and Extranet, and local and distributed computer processingsystems. Storage modules also include, but are not limited to: magneticstorage media, such as floppy discs, hard disc storage media, magnetictape, optical storage media such as CD-ROM, DVD, electronic storagemedia such as RAM, ROM, EPROM, EEPROM and the like, general hard disksand hybrids of these categories such as magnetic/optical storage media.The storage module is adapted or configured for having recorded thereon,for example, sample name, alleleic variants, and frequency of eachalleleic variant. Such information may be provided in digital form thatcan be transmitted and read electronically, e.g., via the Internet, ondiskette, via USB (universal serial bus) or via any other suitable modeof communication.

As used herein, “stored” refers to a process for encoding information onthe storage module. Those skilled in the art can readily adopt any ofthe presently known methods for recording information on known media togenerate manufactures comprising expression level information.

In one embodiment of any of the systems described herein, the storagemodule stores the output data from the determination module. Inadditional embodiments, the storage module stores the referenceinformation such as expression levels of the marker genes describedherein in subjects who do not have symptoms associated with emphysema.In certain embodiments, the storage module stores the referenceinformation such as expression levels of the marker genes describedherein in a sample of healthy lung tissue obtained from the subject orin a sample from the subject taken at an earlier time.

The “computing module” can use a variety of available software programsand formats for computing the relative expression level of the markergenes described herein. Such algorithms are well established in the art.A skilled artisan is readily able to determine the appropriatealgorithms based on the size and quality of the sample and type of data.The data analysis tools described in Examples can be implemented in thecomputing module of the invention. In one embodiment, the computingmodule further comprises a comparison module, which compares theexpression level of at least two markers genes in the lung tissue sampleobtained from a subject as described herein with the referenceexpression level of those marker genes (FIG. 10). By way of an example,when the expression level of ACVRL1 in the lung tissue sample obtainedfrom a subject is measured, a comparison module can compare or match theoutput data—with the reference expression level of ACVRL1 in a referencesample. In certain embodiments, the reference expression level can havebeen pre-stored in the storage module. During the comparison or matchingprocess, the comparison module can determine whether the expressionlevel in the lung tissue sample obtained from a subject is lower thanthe reference expression level to a statistically significant degree. Invarious embodiments, the comparison module can be configured usingexisting commercially-available or freely-available software forcomparison purpose, and may be optimized for particular data comparisonsthat are conducted.

The computing and/or comparison module, or any other module of theinvention, can include an operating system (e.g., UNIX) on which runs arelational database management system, a World Wide Web application, anda World Wide Web server. World Wide Web application includes theexecutable code necessary for generation of database language statements(e.g., Structured Query Language (SQL) statements). Generally, theexecutables will include embedded SQL statements. In addition, the WorldWide Web application may include a configuration file which containspointers and addresses to the various software entities that comprisethe server as well as the various external and internal databases whichmust be accessed to service user requests. The Configuration file alsodirects requests for server resources to the appropriate hardware—as maybe necessary should the server be distributed over two or more separatecomputers. In one embodiment, the World Wide Web server supports aTCP/IP protocol. Local networks such as this are sometimes referred toas “Intranets.” An advantage of such Intranets is that they allow easycommunication with public domain databases residing on the World WideWeb (e.g., the GenBank or Swiss Pro World Wide Web site). Thus, in aparticular preferred embodiment of the present invention, users candirectly access data (via Hypertext links for example) residing onInternet databases using a HTML interface provided by Web browsers andWeb servers (FIG. 11).

The computing and/or comparison module provides a computer readablecomparison result that can be processed in computer readable form bypredefined criteria, or criteria defined by a user, to provide contentbased in part on the comparison result that may be stored and output asrequested by a user using an output module, e.g., a display module.

In some embodiments, the content displayed on the display module can bethe relative expression levels of at least two marker genes in the lungtissue sample obtained from a subject as compared to a referenceexpression level. In certain embodiments, the content displayed on thedisplay module can indicate whether at least two marker genes were foundto have a statistically significant variation in expression between thelung tissue sample obtained from a subject as compared to a referenceexpression level. In certain embodiments, the content displayed on thedisplay module can indicate the degree to which at least two markergenes were found to have a statistically significant variation inexpression between the lung tissue sample obtained from a subject ascompared to a reference expression level. In certain embodiments, thecontent displayed on the display module can indicate whether the subjecthas an increased risk of having emphysema. In certain embodiments, thecontent displayed on the display module can indicate whether the subjectis in need of a treatment for emphysema. In certain embodiments, thecontent displayed on the display module can indicate whether the subjecthas an increased risk of having a more severe case of emphysema. In someembodiments, the content displayed on the display module can be anumerical value indicating one of these risk or probabilities. In suchembodiments, the probability can be expressed in percentages or afraction. For example, higher percentage or a fraction closer to 1indicates a higher likelihood of a subject having emphysema. In someembodiments, the content displayed on the display module can be singleword or phrases to qualitatively indicate a risk or probability. Forexample, a word “unlikely” can be used to indicate a lower risk forhaving emphysema, while “likely” can be used to indicate a high risk forhaving emphysema.

In one embodiment of the invention, the content based on the computingand/or comparison result is displayed on a computer monitor. In oneembodiment of the invention, the content based on the computing and/orcomparison result is displayed through printable media. The displaymodule can be any suitable device configured to receive from a computerand display computer readable information to a user. Non-limitingexamples include, for example, general-purpose computers such as thosebased on Intel PENTIUM-type processor, Motorola PowerPC, Sun UltraSPARC,Hewlett-Packard PA-RISC processors, any of a variety of processorsavailable from Advanced Micro Devices (AMD) of Sunnyvale, Calif., or anyother type of processor, visual display devices such as flat paneldisplays, cathode ray tubes and the like, as well as computer printersof various types.

In one embodiment, a World Wide Web browser is used for providing a userinterface for display of the content based on the computing/comparisonresult. It should be understood that other modules of the invention canbe adapted to have a web browser interface. Through the Web browser, auser can construct requests for retrieving data from thecomputing/comparison module. Thus, the user will typically point andclick to user interface elements such as buttons, pull down menus,scroll bars and the like conventionally employed in graphical userinterfaces.

Systems and computer readable media described herein are merelyillustrative embodiments of the invention for assessing the state of thelungs of subject by measuring the expression level of at least two ofthe marker genes described herein, and therefore are not intended tolimit the scope of the invention. Variations of the systems and computerreadable media described herein are possible and are intended to fallwithin the scope of the invention.

The modules of the machine, or those used in the computer readablemedium, may assume numerous configurations. For example, function may beprovided on a single machine or distributed over multiple machines.

Lung Tissue Sample

Provided herein are methods, assays and systems for assessing the stateof the lungs of a subject by measuring the expression level of at leasttwo marker genes as described herein in the lung tissue sample obtainedfrom a subject. The term “lung tissue sample” as used herein denotes asample taken or isolated from a biological organism, e.g., lung biopsysample, tissue cell culture supernatant, cell lysate, a homogenate of atissue sample from a subject or a fluid sample from a subject. Exemplarybiological samples include, but are not limited to, lung biopsies, theexternal sections of the respiratory tract, lung epithelial cells, etc.The term also includes both a mixture of the above-mentioned samples.The term “lung tissue sample” also includes untreated or pretreated (orpre-processed) biological samples. A lung tissue sample can containcells from subject, but the term can also refer to non-cellularbiological material, such as non-cellular fractions that can be used tomeasure gene expression levels. In some embodiments, the sample is froma resection, biopsy, or core needle biopsy. In addition, fine needleaspirate samples can be used. Samples can be either paraffin-embedded orfrozen tissue.

The sample can be obtained by removing a sample of cells from a subject,but can also be accomplished by using previously isolated cells (e.g.isolated at a prior timepoint and isolated by the same or anotherperson). In addition, the lung tissue sample can be freshly collected ora previously collected sample. Furthermore, the lung tissue sample canbe utilized for the detection of the presence and/or quantitative levelof a gene expression product of marker genes as described herein. Insome embodiments, a maker gene expression product is a biomolecule.Representative biomolecules include, but are not limited to, DNA, RNA,mRNA, polypeptides, and derivatives and fragments thereof. In someembodiments, the lung tissue sample can be used for expression analysisfor diagnosis of a disease or a disorder, e.g., emphysema, using themethods, assays and systems of the invention.

In some embodiments, lung tissue sample is a biological fluid. Examplesof biological fluids include, but are not limited to, saliva, blood,sputum, an aspirate, and any combinations thereof.

In some embodiments, the lung tissue sample is an untreated lung tissuesample. As used herein, the phrase “untreated lung tissue sample” refersto a lung tissue sample that has not had any prior sample pre-treatmentexcept for dilution and/or suspension in a solution. Exemplary methodsfor treating a lung tissue sample include, but are not limited to,centrifugation, filtration, sonication, homogenization, heating,freezing and thawing, and any combinations thereof.

In some embodiments, the lung tissue sample is a frozen lung tissuesample, e.g., a frozen tissue or fluid sample such as sputum. The frozensample can be thawed before employing methods, assays and systems of theinvention. After thawing, a frozen sample can be centrifuged beforebeing subjected to methods, assays and systems of the invention.

In some embodiments, the lung tissue sample can be treated with at leastone chemical reagent, such as a protease inhibitor. In some embodiments,the lung tissue sample is a clarified lung tissue sample, for example,by centrifugation and collection of a supernatant comprising theclarified lung tissue sample.

In some embodiments, a lung tissue sample is a pre-processed lung tissuesample, for example, supernatant or filtrate resulting from a treatmentselected from the group consisting of centrifugation, filtration,sonication, homogenization, lysis, thawing, amplification, purification,restriction enzyme digestion ligation and any combinations thereof. Insome embodiments, a lung tissue sample can be a nucleic acid productamplified after polymerase chain reaction (PCR). The term “nucleic acid”used herein refers to DNA, RNA, or mRNA.

In some embodiments, the lung tissue sample can be treated with achemical and/or biological reagent. Chemical and/or biological reagentscan be employed to protect and/or maintain the stability of the sample,including biomolecules (e.g., nucleic acid and protein) therein, duringprocessing. One exemplary reagent is a protease inhibitor, which isgenerally used to protect or maintain the stability of protein duringprocessing. In addition, or alternatively, chemical and/or biologicalreagents can be employed to release nucleic acid or protein from thesample.

The skilled artisan is well aware of methods and processes appropriatefor pre-processing of biological samples required for determination ofexpression of gene expression products as described herein.

Assays for Identifying Compounds Useful in Treating COPD and/orEmphysema

As described herein, the inventors have identified certain genes whichare upregulated or downregulated to a statistically significant degreein tissue which is subject to emphysematous tissue damage as compared toa reference sample. Accordingly, some embodiments of the invention aregenerally related to assays, methods and systems for assessing whether acompound can be useful in treating or preventing the progression ofCOPD, emphysema and/or emphysematous damage (i.e. it causes generegulation changes opposed to those caused by emphysematous tissuedamage or prevents the gene regulation changes caused by emphysematoustissue damage).

In certain embodiments, the assays, methods and systems relate toidentifying a compound which causes a change in the expression level oftwo or more marker genes in a healthy biological sample (i.e. a samplenot exhibiting any signs or symptoms of emphysematous damage, orobtained from a patient not diagnosed with emphysema and/or COPD, etc)which is opposed to the type of change (i.e. upregulation ordownregulation) observed in tissue suffering from emphysematous damage,as described elsewhere herein. In certain embodiments, the expressionlevel of a gene product of at least two marker genes in a biologicalsample of a subject, i.e. at least two marker genes, or at least threemarker genes, or at least four marker genes or at least five markergenes etc, selected from any in a combination of Table 1 and Table 2 asdescribed herein, in response to a compound are determined. In someembodiments, the marker genes are selected from the group consisting ofITGB1, NEDD9, ACVRL1, SMAD6 and TGFBR2. In certain embodiments, theassays, methods and systems are directed to determination of theexpression level of a gene product (e.g. protein and/or gene transcriptsuch as mRNA) in a biological sample which has been treated with acompound.

In some embodiments, the methods and assays described herein include (a)transforming the gene expression product into a detectable gene target;(b) measuring the amount of the detectable gene target; and (c)comparing the amount of the detectable gene target to an amount of areference, wherein if the amount of the detectable gene target isstatistically different from that of the amount of the reference level,and the type of difference in expression level (i.e. upregulation ordownregulation) is opposed to that observed for tissue suffering fromemphysematous damage versus a reference level, the compound isidentified as being useful in the treatment of COPD and/or emphysema.

In certain embodiments, the assays, methods and systems relate toidentifying a compound which prevents, decreases, or reverses the changein the expression level of two or more marker genes which is observed ina biological sample suffering from emphysematous damage. In certainembodiments, the expression level of a gene product of at least twomarker genes in a biological sample of a subject, i.e. at least twomarker genes, or at least four marker genes, or at least five markergenes, or at least eight marker genes, or at least ten marker genes, orat least fifteen marker genes or at least twenty marker genes etc,selected from any in a combination of Table 1 and Table 2 as describedherein, in response to a compound are determined. In some embodiments,the marker genes are selected from a combination of the genes from Table1 and Table 2, for example, at least two genes from Table 1 and at leasttwo genes from Table 2. In one embodiment, the genes are from the groupconsisting of ITGB1, NEDD9, ACVRL1, SMAD6 and TGFBR2. In certainembodiments, the assays, methods and systems are directed todetermination of the expression level of a gene product (e.g. proteinand/or gene transcript such as mRNA) in a biological sample which hasbeen treated with a compound.

In some embodiments, the methods and assays described herein include (a)transforming the gene expression product in 1) a sample suffering fromemphysematous damage and contacted with a compound and 2) a samplesuffering from emphysematous damage into detectable gene targets; (b)measuring the amount of the detectable gene targets; and (c) comparingthe amount of the detectable gene targets to an amount of a referencetissue, wherein if the amount of the detectable gene target in thesample contacted with the compound is less statistically different fromthat of the amount of the reference level than the amount of thedetectable gene target in the sample not contacted with the compound,the compound is identified as being useful in the treatment of COPDand/or emphysema.

In some embodiments, the reference can be a level of expression of themarker gene product in a normal healthy subject with no symptoms orsigns of emphysema. For example, a normal healthy subject has normallung function, and/or is not diagnosed with emphysema and/or COPD,and/or has no history of smoking tobacco, and/or has not been exposed toasbestos or other environmental factors known to contribute to emphysema(e.g. smoke, dust, fires, etc.). In some embodiments, the reference canalso be a level of expression in cultured cells or cultured lung cells.In some embodiments, the reference can also be a level of expression ofthe marker gene product in a control sample, a pooled sample of controlindividuals or samples or a numeric value or range of values based onthe same. In some embodiments, the reference can also be a level ofexpression of the marker gene product in a tissue sample taken fromundiseased lung tissue of a subject. In certain embodiments, wherein theprogression of emphysematous tissue damage in a sample is to bemonitored over time, the reference can also be a level of expression ofthe marker gene product in a tissue sample taken at an earlier date.

In certain embodiments, the marker gene(s) are selected from the geneslisted in Table 1 and/or Table 2. In certain embodiments, the markergenes are selected from any in a combination of Table 1 and Table 2. Insome embodiments, the marker genes are selected from a combination ofthe genes from Table 1 and Table 2, for example, at least two genes fromTable 1 and at least two genes from Table 2. In one embodiment, thegenes are from the group consisting of ITGB1, NEDD9, ACVRL1, SMAD6 andTGFBR2.

In some embodiments, the methods for measuring gene expression are asdescribed elsewhere herein. In some embodiments, there are providedsystems for performing the assays described herein. Examples of suchsystems are described in detail elsewhere herein.

Biological Sample

As used in the context of assays, methods, and systems to identifycompounds useful in treating or preventing COPD, emphysema and/oremphysematous damage the term “biological sample” as used herein denotesa sample of taken or isolated from 1) a biological organism, e.g., lungbiopsy sample, tissue cell culture supernatant, cell lysate, ahomogenate of a tissue sample from a subject or a fluid sample from asubject or 2) an ex vivo cell, tissue, or population of cells, e.g.cells, a cell, tissue culture supernatant, lysate cell lysate,homogenized tissue, etc. Exemplary biological samples include, but arenot limited to, lung biopsies, the external sections of the respiratorytract, lung epithelial cells, cultured lung epithelial cells, etc. Theterm also includes a mixture of the above-mentioned samples. The term“biological sample” also includes untreated or pretreated (orpre-processed) biological samples. A biological sample can containcells, but the term can also refer to non-cellular biological material,such as non-cellular fractions that can be used to measure geneexpression levels. In some embodiments, the sample is from a resection,biopsy, or core needle biopsy. In addition, fine needle aspirate samplescan be used. Samples can be either paraffin-embedded or frozen tissue.

The sample can be obtained by removing a sample of cells from a subject,but can also be accomplished by using previously isolated cells (e.g.isolated at a prior timepoint and isolated by the same or anotherperson). In addition, the biological sample can be freshly collected ora previously collected sample. Furthermore, the biological sample can beutilized for the detection of the presence and/or quantitative level ofa gene expression product of marker genes as described herein. In someembodiments, a maker gene expression product is a biomolecule.Representative biomolecules include, but are not limited to, DNA, RNA,mRNA, polypeptides, and derivatives and fragments thereof.

In some embodiments, biological sample is a biological fluid. Examplesof biological fluids include, but are not limited to, saliva, blood,sputum, an aspirate, and any combinations thereof.

In some embodiments, the biological sample is an untreated lung tissuesample. As used herein, the phrase “untreated biological sample” refersto a biological sample that has not had any prior sample pre-treatmentexcept for dilution and/or suspension in a solution. Exemplary methodsfor treating a biological sample include, but are not limited to,centrifugation, filtration, sonication, homogenization, heating,freezing and thawing, and any combinations thereof.

In some embodiments, the biological sample is a frozen biologicalsample, e.g., a frozen tissue or fluid sample such as sputum. The frozensample can be thawed before employing methods, assays and systems of theinvention. After thawing, a frozen sample can be centrifuged beforebeing subjected to methods, assays and systems of the invention.

In some embodiments, the biological sample can be treated with at leastone chemical reagent, such as a protease inhibitor. In some embodiments,the biological sample is a clarified biological sample, for example, bycentrifugation and collection of a supernatant comprising the clarifiedbiological sample.

In some embodiments, a biological sample is a pre-processed biologicalsample, for example, supernatant or filtrate resulting from a treatmentselected from the group consisting of centrifugation, filtration,sonication, homogenization, lysis, thawing, amplification, purification,restriction enzyme digestion ligation and any combinations thereof. Insome embodiments, a biological sample can be a nucleic acid productamplified after polymerase chain reaction (PCR). The term “nucleic acid”used herein refers to DNA, RNA, or mRNA.

In some embodiments, the biological sample can be treated with achemical and/or biological reagent. Chemical and/or biological reagentscan be employed to protect and/or maintain the stability of the sample,including biomolecules (e.g., nucleic acid and protein) therein, duringprocessing. One exemplary reagent is a protease inhibitor, which isgenerally used to protect or maintain the stability of protein duringprocessing. In addition, or alternatively, chemical and/or biologicalreagents can be employed to release nucleic acid or protein from thesample.

The skilled artisan is well aware of methods and processes appropriatefor pre-processing of biological samples required for determination ofexpression of gene expression products as described herein.

Contacting a Biological Sample with a Compound

Provided herein, assays, methods and systems for assessing whether acompound can be useful in treating or preventing the progression ofCOPD, emphysema and/or emphysematous damage. In some embodiments, theseaspects of the invention involve contacting a biological sample with acompound. A biological sample can be contacted with a compound at anytime prior to transforming the gene expression product into a detectablegene target. For example, the biological sample can be contacted withthe compound 1 minute prior to transformation, 30 minutes prior totransformation, 1 hour prior to transformation, 12 hours prior totransformation, 1 day prior to transformation, 1 week prior totransformation, 1 month prior to transformation, or more.

In some embodiments, a biological sample can be contacted with acompound once. In some embodiments, a biological sample can be contactedwith a compound repeatedly. In some embodiments, a biological sample canbe contacted with a combination of two or more compounds. In someembodiments, one or more compounds can be compounds which have not beenidentified as useful in treating COPD, emphysema and/or emphysematousdamage and one or more compounds can be compounds which have previouslybeen identified as useful or used to treat COPD, emphysema, and/oremphysematous damage.

In some embodiments, a subject can be contacted with a compound and abiological sample is subsequently obtained from the subject for use in aassay, method, or system as described herein. In some embodiments, abiological sample is obtained from a subject and subsequently contactedwith a compound. In some embodiments, the biological sample contactedwith a compound is not obtained directly from a subject, e.g. thebiological sample comprises cultured cells.

In some embodiments, a compound can be a hormone, enzyme, cell, genesilencing molecule, inhibitor of an enzyme, small molecule, peptide,protein, nucleotide, antibody, antibody fragment, growth factor, virus,and/or bacterium.

Some embodiments of the present invention can be defined as any of thefollowing numbered paragraphs.

What is claimed herein is:

-   1. A method for treating chronic obstructive pulmonary disorder    (COPD) or emphysema in a subject by administering to the subject a    composition comprising a GHK tripeptide.-   2. A method for treating chronic obstructive pulmonary disorder    (COPD) or emphysema in a subject comprising:    -   (a) determining if a subject has or is at risk for COPD or        emphysema; and    -   (b) administering an effective amount of a composition        comprising a GHK tripeptide if the individual is positive in        step (a).-   3. The method of Paragraph 1, further comprising performing an assay    prior to administering the GHK tripeptide, the assay comprising;    -   subjecting a test sample of a subject to at least one analysis        to determine the level of expression of at least 2 marker gene        products from at least 1 of the following 2 groups,    -   Group A, wherein Group A is selected from the group consisting        of:        -   SEQ ID NOs: 1-79, 128-205, 253-316, and 379-442.    -   Group B, wherein Group B is selected from the group consisting        of:        -   SEQ ID NOs: 80-127, 206-252, 317-378, and 443-504.    -   wherein an expression profile of 2 or more marker gene products        of Group A which is decreased relative to a reference level and        an expression profile of 2 or more marker gene products of Group        B which is increased relative to a reference level, indicates        the presence of emphysema.-   4. The method of Paragraph 4, further comprising administering the    GHK tripeptide if the presence of emphysema is indicated.-   5. The method of Paragraph 4, further comprising not administering    the GHK tripeptide if the presence of emphysema is not indicated.-   6. The use of a GHK peptide in the manufacture of a medicament for    the treatment of emphysema or COPD.-   7. The use of a pharmaceutical composition comprising a GHK    tripeptide for the treatment of COPD or emphysema.-   8. A method of reversing emphysematous lung destruction in a subject    by contacting the lung with a GHK tripeptide.-   9. The method or use of any of paragraphs 1-8, wherein the    composition or medicament further comprises a pharmaceutically    acceptable carrier.-   10. The method or use of any of paragraphs 1-9, wherein the    composition or medicament is administered to the airspace of the    lung.-   11. The method or use of paragraph 10, wherein the composition or    medicament is administered orally or nasally.-   12. The method or use of paragraph 10-11 wherein the composition or    medicament is administered using an inhaler or a nebulizer.-   13. The method or use of any of paragraphs 1-12, wherein the GHK is    not complexed with copper.-   14. The method or use of any of paragraphs 1-13 wherein the subject    is a mammal.-   15. The method or use of paragraph 14, wherein the subject is a    human.-   16. The method or use of any of paragraphs 1-14, wherein the subject    is at risk of developing COPD.-   17. The method or use of any of paragraphs 1-16, wherein the method    further comprises selecting a subject in need of reversal of    emphysematous lung destruction prior to contacting the lung tissue    of the subject with the composition.-   18. The method or use of any of paragraphs 1-17, wherein the subject    smokes tobacco.-   19. The method or use of any of paragraphs 1-18, wherein the subject    has been exposed to asbestos, air pollution, or environmental    hazards such as dust, chemicals, fires, and/or smoke.-   20. The method or use of any of paragraphs 1-19, wherein the subject    has low levels of a-1 antitrypsin (AAT) in the blood.-   21. The method or use of any of paragraphs 1-20, wherein the subject    is administered an additional treatment for emphysema or COPD.-   22. The method or use of any of Paragraphs 1-21, wherein the    additional treatment for emphysema or COPD is selected from the    group consisting of:    -   a bronchodilator; albuterol; ipratropium bromide;        methylxanthine; steroids; antibiotics; and oxygen.-   23. An assay for assessing the state of the lungs of a subject, the    assay comprising:    -   subjecting a test sample of a subject to at least one analysis        to determine the level of expression of at least 2 marker gene        products from at least 1 of the following 2 groups,    -   Group A, wherein Group A is selected from the group consisting        of:        -   SEQ ID NOs: 1-79, 128-205, 253-316, and 379-442.    -   Group B, wherein Group B is selected from the group consisting        of:        -   SEQ ID NOs: 80-127, 206-252, 317-378, and 443-504.    -   wherein an expression profile of 2 or more marker gene products        of Group A which is decreased relative to a reference level and        an expression profile of 2 or more marker gene products of Group        B which is increased relative to a reference level, indicates        the presence of emphysema.-   24. The assay of Paragraph 23, wherein the expression profile is    determined for at least three gene products of Group A and at least    three gene products from Group B.-   25. The assay of any of Paragraphs 23-24, wherein the expression    profile is determined for at least 2 of the following 5 genes;    -   ITGB1, NEFF9, ACVRL1, SMAD6 and TGFBR2.-   26. The assay of any of Paragraphs 23-25, wherein the expression    profile is determined for at least 3 of the following 5 genes;    -   ITGB1, NEFF9, ACVRL1, SMAD6 and TGFBR2.-   27. The assay of any of Paragraphs 23-26, wherein the expression    profile is determined for at least 4 of the following 5 genes;    -   ITGB1, NEFF9, ACVRL1, SMAD6 and TGFBR2.-   28. The assay of any of Paragraphs 23-27, wherein the expression    profile is determined for the following 5 genes;    -   ITGB1, NEFF9, ACVRL1, SMAD6 and TGFBR2.-   29. The assay of any of Paragraphs 23-28, further comprising;    -   wherein the subject is administered a treatment for emphysema or        COPD if the expression profile of 2 or more marker gene products        of Group A is decreased relative to a reference level and the        expression profile of 2 or more marker gene products of Group B        is increased relative to a reference level; or    -   wherein the subject is not administered a treatment for        emphysema or COPD if the expression profile of 2 or more marker        gene products of Group A is not decreased relative to a        reference level and the expression profile of 2 or more marker        gene products of Group B is not increased relative to a        reference level.-   30. The assay of Paragraph 29, wherein the treatment for emphysema    or COPD is selected from the group consisting of:    -   a GHK tripeptide; a bronchodilator; albuterol; ipratropium        bromide; methylxanthine; steroids; antibiotics; and oxygen.-   31. The assay of Paragraph 29, wherein the treatment for emphysema    or COPD comprises administering a GHK tripeptide according the    methods of any of paragraphs 1-22.-   32. The assay of any of Paragraphs 23-28, wherein a statistically    significant difference in expression levels of at least two marker    genes beyond a predetermined threshold identifies a subject with    severe emphysema.-   33. The assay of any of Paragraphs 23-28 and 32, wherein a subject    indicated to have emphysema present in their lungs is administered a    GHK tripeptide according to the methods of any of paragraphs 1-22.-   34. The assay of any of Paragraphs 23-33, wherein the level of    expression of a marker gene product is determined using an method    selected from the group consisting of:    -   RT-PCR; quantitative RT-PCR; Northern blot; microarray based        expression analysis; Western blot; immunoprecipitation;        enzyme-linked immunosorbent assay (ELISA); radioimmunological        assay (RIA); sandwich assay; fluorescence in situ hybridization        (FISH); immunohistological staining; radioimmunometric assay;        immunofluoresence assay; mass spectroscopy and        immunoelectrophoresis assay.-   35. A computer system for detecting emphysema in subject, the system    comprising:    -   a determination module configured to identify and detect the        expression level of at least four marker gene products in a lung        tissue sample obtained from a subject, wherein at least two        marker gene products are selected from the group consisting of        SEQ ID NOs: 1-79, 128-205, 253-316, and 379-442and at least two        marker gene products are selected from the group consisting of        SEQ ID NOs: 80-127, 206-252, 317-378, and 443-504;    -   a storage module configured to store output data from the        determination module;    -   a comparison module adapted to identify from the output data        whether the overall level of expression of the at least four        marker genes in the lung tissue sample obtained from a subject        varies by a statistically significant amount from the expression        level found in a reference sample;    -   a display module for displaying whether two or more marker gene        products have a statistically significant variation in        expression level in the lung tissue sample obtained from a        subject as compared to the reference expression level and/or        displaying the relative expression levels of the marker gene        products.-   36. The system of paragraph 35 wherein two or more of the marker    genes is selected from the group consisting of ITGB1, NEFF9, ACVRL1,    SMAD6 and TGFBR2.-   37. The system of any of paragraphs 35-36, wherein if the computing    module determines that the overall expression level of the at least    four marker genes in the lung tissue sample obtained from a subject    varies by a statistically significant amount from the reference    expression level, the display module displays a signal indicating    that the expression levels in the sample obtained from a subject    vary from those of the reference expression level.-   38. The system of any of paragraphs 35-37, wherein the signal    indicates that the subject has an increased likelihood of having    emphysema.-   39. The system of any of paragraphs 35-38, wherein the signal    indicates the subject is in need of treatment for emphysema.-   40. The system of any of paragraphs 35-39, wherein the signal    indicates the degree to which the expression level of the marker    genes in the sample obtained from a subject vary from the reference    expression level.-   41. The system of any of paragraph 35-40, wherein the signal    indicates that the subject has an increased likelihood of having a    more severe case of emphysema.-   42. An assay for identifying a compound which can be used in    treating COPD and/or emphysema, the assay comprising:    -   using a biological sample containing at least four marker gene        products, wherein at least two of the gene products are from the        group consisting of SEQ ID NOs: 1-504;    -   measuring the expression level of the marker genes;    -   comparing the expression level of the marker genes from the        biological sample treated with the compound to reference levels        the marker genes;    -   wherein the reference levels are obtained from a biological        sample not treated with the compound;    -   wherein an overall statistically significant difference in the        expression levels of the four marker genes in the biological        sample treated with the compound relative to the reference        levels indicates the compound can be used in treating COPD        and/or emphysema.

The present invention is further illustrated by the following exampleswhich in no way should be construed as being further limiting, Thecontents of all cited references, including literature references,issued patents, published patent applications, and co-pending patentapplications, cited throughout this application are hereby expresslyincorporated by reference.

The present invention has been described in terms of particularembodiments found or proposed by the present inventor to comprisepreferred modes for the practice of the invention. It will beappreciated by those of skill in the art that, in light of the presentdisclosure, numerous modifications and changes can be made in theparticular embodiments exemplified without departing from the intendedscope of the invention. For example, due to codon redundancy, changescan be made in the underlying DNA sequence without affecting the proteinsequence. Moreover, due to biological functional equivalencyconsiderations, changes can be made in protein structure withoutaffecting the biological action in kind or amount. All suchmodifications are intended to be included within the scope of theappended claims.

EXAMPLES

Described herein are experiments which correlate gene expression changeswith the degree of emphysematous lung damage caused by COPD and/oremphysema. These studies have identified marker genes which are subjectto statistically significant up-or down-regulation in lung tissuesuffering emphysematous damage as compared to undamaged or less damagedtissue. Also described herein is work which identifies GHK as a compoundwhich induces gene expression changes which are the opposite of thoseobserved in tissue suffering from emphysematous lung damage. As shownherein, GHK causes these gene expression changes in human lungfibroblasts and is identified as a compound for treating COPD and/oremphysema and/or reversing emphysematous lung damage.

Example 1 Materials and Methods

Sample Acquisition and Processing

Single lungs (n=6) were removed from patients treated for severe COPD bydouble lung transplantation at the University of Pennsylvania and donorlungs (n=2) for which no suitable recipient was identified were releasedfrom the Gift of Life Organ Procurement Organization in Philadelphia.Each lung was removed from the thorax, cooled to 1.6° C., andtransported to the laboratory where the bronchial stump was cannulated(Choong, et al., J Thorac Cardiovasc Surg 2005 130: 922-3). The lung wasthen inflated using a compressed air source attached to an underwaterseal to slowly increase transpulmonary pressure (PL) from 0 to 30 cmH₂O. The specimen was then held at PL of 10 cm H₂O while frozen byliquid nitrogen vapor (−130° C.). The frozen specimen had a MDCT scanand followed by being cut into 2 cm thick slices in the same plane asthe CT scan. Tissue samples were collected using a sharpened steelcylinder (cork bore diameter: 14 mm). One sample from a cluster of fourcore samples of lung obtained from each site was processed for micro CTa companion core was used for the gene profiling and validation studiesreported here.

The severity of emphysema within each core was estimated by measuringthe mean linear intercept (Lm) by micro-CT. The micro-CT scan of each 2cm long core provided approximately 1000 contiguous 4 μm thick images.Lm was measured from these images at 20 regular intervals along the 2 cmlength of the core.

Measurement of Mean Linear Intercept

Mean linear intercept (Lm) was measured at 20 regularly spaced intervalsof each of the microCT scans of lung samples adjacent to samples usedfor gene expression using a previously validated grid of test linesprojected onto the image and a custom macro linked to specializedsoftware (ImagePro Plus; MediaCybernetics, Silver Spring, Md., USA). Agrid of test lines of a known length was applied onto the image. Thenumber of intercepts between these lines and tissue was counted with Lmcalculated as the total length of the test lines divided by the numberof cross-overs with tissue (equal to the number of intercepts divided by2).

Microarray Sample Processing

High molecular weight (Hmw; mRNA-containing fraction) RNA was isolatedfrom tissue cores using the miRNeasy Mini Kit (Qiagen). The RNAintegrity was assessed using an Agilent 2100 Bioanalyzer and RNA puritywas assessed using a NanoDrop spectrophotometer. One μg of RNA wasprocessed and hybridized onto the Human Exon 1.0 ST array (AffymetrixInc., Santa Clara, Calif.) according to the manufacturer's protocol aspreviously described (Zhang, X. et al. Genome informatics. InternationalConference on Genome Informatics 2007 18:247-57).

Data Preprocessing

Expression Console Version 1.0 (Affymetrix Inc., Santa Clara, Calif.)was used to generate transcript-level gene expression estimates for the“core” exon probe sets via the robust multichip average (RMA) algorithm.Gene symbols of transcript ids were retrieved using DAVID(http://david.abcc.ncifcrf.gov/)28. These gene expression data arefreely available through the Gene Expression Omnibus (GEO) (available atthe world wide web address, ncbi.nlm.nih.gov/geo/) under the accessionGSE27597.

Microarray Data Analysis

Linear mixed-effects models were used to identify gene expressionprofiles associated with the degree of regional emphysema severity asmeasured by Lm while correcting for the slice in the lung from which thecore was taken and treating differences between patients as randomeffects.

Microarray and Immunohistochemistry Data Analysis

Two linear mixed-effects models were used to identify gene expressionprofiles associated with the degree of regional emphysema severity asmeasured by Lm:

Gene_(ij)=β₀+β_(Slice)*Slice_(i)+α+_(j)+ε_(ij)   (1)

Gene_(ij)=β₀+β_(Slice)*Slice_(i)+β_(Lm)*Lm_(i)+α_(j)+ε_(ij)   (2)

i=1,2, . . . ,8; j=1,2, . . . ,8

ε_(ij)˜N(0, σ²) α_(j)˜N(0, σ² _(aj))

Gene_(ij) is the log2 expression value for sample i in patient j for asingle gene. Slice is a fixed effect controlling for the position withinthe lung from which the sample core was obtained. The random term ε_(ij)represents the random error which was assumed to be normallydistributed, α_(j) represents the random effect for patient, and β₀represents the intercept. The model in equation (2) contains anadditional fixed effect term for emphysema severity measured by thenatural log of Lm. A gene's expression profile was considered associatedwith Lm if model (2) fit better than model (1) as determined by asignificant p-value from an ANOVA between the two models after applyinga false discovery rate (FDR) correction. In the immunohistochemistryexperiments, these linear models were also used to examine therelationship between Lm and the volume fraction of tissue with positivestaining by substituting volume fraction (Vv) for gene expression as adependent variable.

Reverse Engineering of Transcriptional Networks

CLR scores between all possible pairs of genes were computed using CLRversion 1.2, and a significance cut-off of FDR q-value <2.0×10⁻⁵ wasselected. The CLR algorithm calculates these scores by computing mutualinformation between all possible pairs of genes and then applying az-score correction. A sub-network was created consisting of edgesbetween differentially expressed transcription factors and genesconnected to these transcription factors and edges betweennon-differentially expressed transcription factors connected todifferentially expressed genes (FIGS. 2A-2C). The direction of thecorrelation was determined by the sign of a Pearson correlation.

Connecting to Other Datasets

GSEA was used to explore connections to other datasets. Genes in thisdataset were ranked by the t-statistic of the Lm term in the linearmodel represented by equation (2) shown above. This list was analyzedfor enrichment of gene sets composed of genes reported to be associatedwith COPD or emphysema in five other studies (Golpon, H. A. et al.American journal of respiratory cell and molecular biology 200431:595-600: Ning, W. et al. PNAS 2004 101:14895-900; Bhattacharya, S. etal. American journal of respiratory cell and molecular biology 200940:359-67; Spira, A. et al. American journal of respiratory cell andmolecular biology 2004 31: 601-10; Wang, I.-M. et al. American journalof respiratory and critical care medicine 2008 177:402-11). Conversely,gene sets consisting of the genes we identified to be significantlyassociated with Lm (FDR <0.1) were used to explore other COPD-relateddatasets with data available from GEO: GSE11223, GSE85007, GSE85815, andGSE16506. For those datasets where raw data was available, the Entrezgene chip definition file (CDF) was used to obtain RMA normalizedexpression values. Genes from these datasets were ranked by t-statistic.For dichotomous variables, the t-statistic was calculated from acomparison of expression levels observed in the two groups (e.g. casesand controls). For continuous variables, the t-statistic was from alinear model of expression as a function of the continuous variable(e.g. FEV1). Datasets that measured the effect of TGFβ ligands on geneexpression in various cell types included Chambers et al., (The Americanjournal of pathology 2003162:533-46) Verrecchia et al., (The Journal ofbiological chemistry 2001 276:17058-62), GSE749712, GSE545010,GSE665314, GSE1171013, and GSE172415. Lists of genes that change withTGFβ exposure were taken from the publication associated with themicroarray data. If lists of at least 25 genes could not be obtainedfrom the literature, the raw data was used from GEO and normalized asdescribed above. The 200 genes with the highest fold change and the 200genes with the lowest fold change were used as gene sets and compared toa list of all genes from this dataset ranked by their correlation to Lm.Conversely, sets of genes significantly correlated to Lm were comparedto lists of all genes ranked by fold change between cells treated anduntreated with TGFβ.

Functional Enrichment Analysis

Functional enrichment analysis was performed using DAVID or gene setenrichment analysis (GSEA) (Dennis, G. et al. Genome biology 2003 4:P3;Subramanian, A. et al. PNAS 2005 102:15545-50). For DAVID, functionalenrichment was examined among GO categories, and KEGG and BIOCARTApathways using a modified Fisher exact test and FDR correction. ForGSEA, genes were ranked by the t-statistic of the Lm term in the linearmixed-effects model and then analyzed for the enrichment of canonicalpathways and GeneOntology (GO) term gene sets obtained from MSigDB(available at the world wide web address,broadinstitute.org/gsea/msigdb/index.jsp).

Real Time PCR Validation

Quantitative RT-PCR analysis was used to confirm the expression levelsof select genes. ACVRL1, BCL11A, CCR7, CD79A, CXCL13, EPAS1, FOXF1,KLF13, S100A8, SMAD6, WFDC1, and TAL1 were associated with Lm (FDR <0.1)while GATA2 and TBX3 and were among the most highly connected genes inthe relevance network. Primer sequences for the fourteen genes chosenfor validation were designed with PRIMER EXPRESS software (AppliedBiosystems, Foster City, Calif.). Primer sequences to measure theexpression of housekeeping genes (GAPDH, TBP, YWHAZ) were adopted fromVandesompele et al., Genome Biology 2002 3:RESEARCH0034. The sequencesused for all primers are provided in Supplementary Table 1. RNA samples(2 μg of RNA from the samples used in the microarray analysis) weretreated with TURBO DNA-free (Ambion, Foster City, Calif.), according tothe manufacturer's protocol, to remove contaminating genomic DNA. TotalRNA was reverse-transcribed using random hexamers (Applied Biosystems)and SuperScript II reverse transcriptase (Invitrogen, Carlsbad, Calif.).The resulting first-strand cDNA was diluted with nuclease-free water(Ambion) to 4 ng/μl. PCR amplification mixtures (25 μl) contained 20 ngtemplate cDNA, 12.5 μl of 2× SYBR Green PCR master mix (AppliedBiosystems) and 300 nM forward and reverse primers. Forty cycles ofamplification and data acquisition were carried out in StepOnePlusReal-Time PCR systems (Applied Biosystems).

Threshold determinations were automatically performed by StepOneSoftware (version 2.0.2; Applied Biosystems) for each reaction. Allreal-time PCR experiments were carried out in triplicate on each sample.Data analysis was performed using geNorm. Three genes (GAPDH, TBP,YWHAZ) were used for normalization.

Immunohistochemistry

Portions of a frozen tissue core close to the source of RNA were vacuumembedded in diluted Tissue-Tek O.C.T. compound (Sakura Finetek USA Inc,Torrance, Calif., USA) (50% vol/vol) in PBS containing 10% sucrose keptjust above the freezing point and immediately refrozen on dry ice.Histology sections cut from these frozen blocks were air-dried at roomtemperature overnight and stained with appropriate antibodies (Table 3;RT=room temperature). Each antibody was optimized prior to performing astaining run on an automatic immunostainer (Dako, Mississauga, ON,Canada) using MACH 4 Universal AP Polymer Detection Kit (BioCareMedical, Concord, Calif., USA), and each section was counterstained withhematoxylin. Non-specific IgG substitution for specific antibodiesprovided negative controls. Digital images were captured using a NikonE-800 microscope and the volume fraction of the tissue containingpositively stained cells was determined using Image-Pro Plus software(Media Cybernetics, Bethesda, Md., USA) (Hogg, J. C. et al. The NewEngland journal of medicine 2004 350:2645-53).

TABLE 3 Antibodies Used for Immunohistochemistry Antibody CompanyCatalog No. Host Dilution Fixation CD79a Dako M7050 Mouse 1/50 acetone,RT, 10 min SMAD1 Abnova DP0239 Rabbit 1/400 cold acetone, 5 min SMAD2LifeSpan LS-C39128 Rabbit 1/100 acetone, RT, 10 min Biosciences SMAD6LifeSpan LS-B2065 Rabitt 1/25 10% formalin, RT, 10 min Biosciences

Connectivity Map

The 50 most up and 50 most down-regulated genes that change with severalconditions were used as separate query signatures for the ConnectivityMap (Lamb, J. et al. Science 2006 313:1929-35). These include geneexpression profiles changing as a function of regional emphysemaseverity, expression profiles changing with FEV1, FEV1/FVC, or betweencases vs. controls in Bhattacharya et al., (American journal ofrespiratory cell and molecular biology 2009 40:359-67), expressionprofiles changing between controls vs. emphysema patients or by controlsvs. al-antitrypsin disease in Goplon et al., (American journal ofrespiratory cell and molecular biology 2004 31:595-600), expressionprofiles changing with FEV1, FEV1/FVC, DLCO, or between cases andcontrols in Spira et al., (American journal of respiratory cell andmolecular biology 2004 31: 601-10) and expression profiles changing withFEV1, non-smokers vs. GOLD2, or non-smokers vs. GOLD3 in Wang et al(American journal of respiratory and critical care medicine 2008177:402-11). In addition, gene expression profiles that change with TGFβtreatment from Qin et al., (BMC systems biology 2009 3:73), Classen etal., (Journal of immunology 2007 178:6931-40), Renzoni et al.,(Respiratory research 2004 5:24), Koinuma et al., (Molecular andcellular biology 2009 29:172-86), and Malizia et al., (American journalof physiology. Lung cellular and molecular physiology 2008 295:L451-60)were used as separate query signatures. For each dataset, gene symbolswere mapped to every corresponding probe set id on the Affymetrix U133Aarray in R using the hgu133a.db package to create the query signatures.For comparison of CMap data to in vitro studies, raw data for GHK wasdownloaded from the CMap website and normalized using MAS5.0. Geneexpression profiles were ranked by a paired t-test between treatment andcontrols of different batches.

Fibroblast Cultures

Normal human diploid lung fibroblasts (HFL-1) were obtained from theAmerican Type Culture Collection (Manassas, Va.) and used between 3-5consequent passages after thawing of frozen cells. HFL-1 cells wereplaced in 6-well culture plates (BD Biosciences, Mississauga, ON) at adensity of 1×10⁵ cells per well in complete cell culture medium(CCM,DMEM supplemented with 10% FBS, 2 mM L-glutamine and 1% ofantibiotic solution; Invitrogen, Burlington, ON). Cells were cultured at37° C. and 5% CO2 in CCM before they reached 80% confluence. Cells werequiesced in CCM with 1% FBS overnight. HFL-1 cultures were treated withGly-His-Lys acetate (GHK, Sigma-Aldrich, Oakville, ON) at concentrationsof 100 pM and 10 nM, recombinant human TGFβ1 (10 ng/ml, PeproTech, RockyHill, N.J.) alone or in combination with GHK for 48 hrs. Cells exposedto the CCM with added DMSO served as a vehicle control. Experiments wererepeated 3 times using HFL-1 cells during 3 consequent cell culturepassages. Total RNA was isolated from HFL-1 monolayers using RNeasy PlusMini-Kit (Qiagen, Valencia, Calif.) as per manufacturer's instructions.Quantity and quality of the isolated RNA was determined using Agilent2100 Bioanalyzer (Agilent Technologies, Mississauga, ON). Isolated RNAwas processed and hybridized onto the Human Gene 1.0 ST array(Affymetrix Inc., Santa Clara, Calif.) according to the manufacturer'sprotocol. Transcript-level gene expression estimates were generatedusing RMA with the Entrez gene CDF version v11. Differentially expressedgene expression profiles were identified using a one-way ANOVA for GHKtreatment and a t-test for TGFβ1 treatment. Gene expression profileswere ranked for each treatment by t-statistic. The 200 most up-regulatedand the 200 most down-regulated gene profiles were used as gene sets foreach treatment in GSEA.

Immunoblotting for β1-Integrin

Production of β1-integrin by HFL-1 cells was assessed by immunoblottingas previously described (Pechkovsky, D. V. et al. The Journal ofbiological chemistry 2008 283:12898-908). Briefly, total cell proteinwas extracted from cultured HFL-1 using Cell Protein Extraction Buffer(Biosource, Camarillo, Calif.), quantified by DC Protein Assay (Bio-Rad,Hercules, Calif.), resolved by SDS-PAGE, and transferred tonitrocellulose membranes. Membranes were probed with mouse anti-humanβ1-integrin antibody (clone 419127, R&D Systems, Minneapolis, Minn.) andanti-β-tubulin antibody (Millipore, Billerica, Mass.) as a loadingcontrol. Detection was performed using the IR800 detectiongoat-anti-mouse antibody (Rockland Immunochemicals, Gilbertsville, Pa.)and Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, Nebr.)as recommended by manufacturer. Density of the bands was quantifiedusing Odyssey software 2.1 (LI-COR Biosciences). The data are presentedas β1 integrin/β-tubulin density ratios.

Statistical Analysis

All statistical analyses were conducted using R statistical software v2.9 and Bioconductor packages v2.4.

Example 2 Expression Profiling of Emphysematous Lung Tissue

Study Population

In order to gain insights into biological processes associated withincreasing emphysema severity and to discover novel compounds for thetreatment of emphysema, paired samples were obtained at 8 regions fromwithin regular intervals between the apex and base of explanted lungsfrom patients with GOLD-4 COPD (n=6) and donors (n=2). The degree ofemphysematous destruction was quantified in one sample from each regionby mean linear intercept (Lm), a morphological measurement of alveolardestruction derived from micro-CT scans of the frozen lung tissuespecimen, while gene expression was profiled in the corresponding samplefrom the same region.

Lm was quantified using micro-CT scans in eight samples from differentregions of lungs from six subjects that required transplantation forCOPD and two organ donors (FIG. 1). Table 4 shows the demographicinformation and clinical characteristics of the eight subjects used inthis study. As expected, samples from subjects with COPD had a highermean and a greater range of Lm values between samples compared to thosefrom donor lungs, indicating that there are regions of severe emphysemain COPD subjects (Table 4). Subject 6967 was diagnosed with a PureAirway Obstruction (PAO) COPD phenotype without emphysema. Reflectingthis diagnosis, the distribution of Lm measurements for this patientclosely resembles the distribution of Lm measurements from the donorlungs. Subject 6970 was diagnosed with α-1 antitrypsin (AAT) deficiency.The remaining four subjects with COPD had the centrilobularemphysematous phenotype commonly observed in smokers. Subject 6969 hadone sample excluded from subsequent analysis because its Lm measurementwas an outlier (more than 3 times the interquartile range of thedistribution of all Lm measurements of all of the lungs examined).Subjects with COPD had FEV1/FVC <70% and FEV1 <25% predicted. Some COPDpatients had other diseases including von Willebrand disease*,hypertension†, and α-1 Antitrypsin deficiency disease‡.

TABLE 4 Subject demographics Lm Lm Patient De- Pack Smoking Mean +/−Range ID scription Sex Age Years Status SD (μm) (μm) 6965 COPD M 62 50Former  716 +/− 494- 164 982 6967 COPD F 61 25 Former 414 +/− 82 334-585 6968 COPD F 63 38 Former  724 +/− 357- 252 1013 6969 COPD*† F 56 54Former 1822 +/− 521- 1270 4620 6970 COPD‡ M 55 15 Former 1352 +/− 647-599 2551 6971 COPD M 59 30 Former 1097 +/− 720- 441 2101 6982 Donor M 59— Never 384 +/− 47 344- 473 6983 Donor M 62 24 Former 289 +/− 41 231-352 von Willebrand disease*; hypertension†; α-1 Antitrypsin deficiencydisease‡

Gene Expression Profiles Associated with Regional Emphysema Severity

By ANOVA, the expression levels of one-hundred twenty-seven genes weresignificantly associated with Lm and thus associated with regionalemphysema severity (Tables 1,2 and 5; FDR <0.10 corresponding to ap-value <0.0007). Table 5 shows data concerning the extent ofdifferential gene expression. The third column of Table 5 shows thecontrolled log2 fold change in gene expression per unit increase in Lm.The fourth column of Table 5 shows the log2 fold change in geneexpression between the healthiest third of the samples used, as measuredby Lm, and the third of the samples most affected by emphysema, asmeasured by Lm.

The expression of 49 genes increased as a function of increasingregional emphysema severity. Genes with functions in the B cell receptorsignaling pathway and the humoral immune response were overrepresentedamong these up-regulated genes (FDR <0.05). In contrast, 78 genes showeddecreased expression as a function of increasing regional emphysemaseverity, and genes involved in cellular structure, integrin signaling,extracellular matrix, focal adhesion, blood vessel morphogenesis, theVEGF pathway, and the TGFβ pathway were over-represented in this list(FDR <0.05; Table 6). The expression of CD79A, a component of the B cellreceptor, increased with increasing emphysema severity (data not shown),and the expression of ACVRL1 (also known as activin-like kinase I), areceptor in the TGFβ pathway, decreased with increasing emphysemaseverity (data not shown). These genes are shown as examples of thecharacteristic relationship between Lm and gene expression observed inthe data provided in Tables 1 and 2. A gene expression relevance networkwas inferred using the Context Likelihood of Related (CLR) algorithm(Faith, J. J. et al. PLoS biology 2007 5:e8) specifically to predicttranscription factors that may play a role in emphysema pathogenesis.Transcription factors with the most edges included EPAS1 (also known asHIF-2α), KLF13, TAL1, TBX3, GATA2, and BCL11A (FIGS. 2A-2C).

TABLE 5 Genes with differential expression in healthy lung tissue andlung tissue with emphysematous damage Log2 Log2 Fold Fold Change/ ChangeT-stat Unit (1^(st) vs. for P-value Increase 3^(rd) Column of ColumnAffy_ID Gene in Lm Tertile) 3 5 FDR 554975 BCL11A 0.29 0.37 7.623.02E−07 0.002696819 3256590 PAPSS2 −0.50 −0.65 −8.15 2.14E−070.002696819 3756319 CCR7 0.46 0.60 6.66 6.12E−07 0.00364382 3671695WFDC1 −0.41 −0.53 −5.98 1.41E−06 0.006309394 3555088 KIAA0125 0.61 0.795.42 2.31E−06 0.008255258 2514216 NOSTRIN −0.77 −0.99 −8.12 6.22E−060.018533994 3709685 NDEL1 −0.25 −0.32 −5.83 9.64E−06 0.024601661 3830359CD22 0.34 0.44 4.63 1.25E−05 0.024783955 3442579 RBP5 0.35 0.45 5.811.17E−05 0.024783955 2452977 FAIM3 0.42 0.54 5.16 1.68E−05 0.0252030513508898 STARD13 −0.30 −0.39 −5.64 1.83E−05 0.025203051 2941784 NEDD9−0.49 −0.63 −5.90 1.83E−05 0.025203051 2514304 DHRS9 0.74 0.95 5.001.73E−05 0.025203051 3333443 ASRGL1 −0.40 −0.52 −5.58 2.57E−050.032786798 3518766 EDNRB −0.57 −0.74 −4.56 2.86E−05 0.034134938 3587015KLF13 −0.24 −0.31 −5.15 3.37E−05 0.037650711 2363852 FCRLA 0.29 0.384.82 4.11E−05 0.03862529 3223928 STOM −0.22 −0.28 −5.24 3.91E−050.03862529 3671727 ATP2C2 0.31 0.40 5.37 4.05E−05 0.03862529 3865503GPR4 −0.41 −0.52 −4.41 4.81E−05 0.042988418 3209384 TMEM2 −0.41 −0.53−4.61 6.24E−05 0.048500471 2610972 SYN2 −0.31 −0.40 −4.30 6.07E−050.048500471 3662158 MT1JP −1.33 −1.71 −5.39 5.71E−05 0.048500471 2553576RTN4 −0.23 −0.29 −4.39 9.35E−05 0.055840204 3142217 PAG1 −0.41 −0.52−6.26 9.16E−05 0.055840204 3501219 COL4A2 −0.31 −0.40 −4.14 8.98E−050.055840204 3665029 CES3 0.19 0.25 5.11 9.02E−05 0.055840204 2640993KLHDC6 0.25 0.32 5.13 8.86E−05 0.055840204 2403080 FCN3 −0.62 −0.80−4.40 9.69E−05 0.055840204 3368940 ABTB2 −0.15 −0.20 −5.66 9.51E−050.055840204 2327219 STX12 −0.34 −0.44 −4.50 8.97E−05 0.055840204 3415109ACVRL1 −0.51 −0.65 −4.89 1.12E−04 0.062276427 2955932 GPR110 0.74 0.964.28 1.29E−04 0.0634364 3102204 C8orf34 0.38 0.49 4.73 1.18E−040.0634364 2438411 NES −0.30 −0.39 −4.42 1.24E−04 0.0634364 2453365PLXNA2 −0.35 −0.45 −4.17 1.31E−04 0.0634364 2377283 CR2 0.44 0.57 4.511.31E−04 0.0634364 3073013 PODXL −0.47 −0.60 −4.76 1.39E−04 0.0655837463766796 PECAM1 −0.36 −0.46 −4.59 1.49E−04 0.066237807 3094778 TACC1−0.27 −0.34 −5.61 1.59E−04 0.066237807 3486956 C13orf15 −0.43 −0.56−4.11 1.56E−04 0.066237807 2435989 S100A8 −0.96 −1.24 −4.01 1.58E−040.066237807 2876543 C5orf20 0.22 0.28 5.11 1.57E−04 0.066237807 2509988LOC130576 0.21 0.27 4.01 1.65E−04 0.067004025 3590129 ZFYVE19 0.12 0.164.09 1.69E−04 0.067228391 3226097 ENG −0.33 −0.43 −4.46 1.75E−040.067825806 3955915 TPST2 −0.17 −0.22 −4.00 1.80E−04 0.068624866 3629652NOPE 0.16 0.21 4.16 1.91E−04 0.069991223 3415320 KRT7 −0.55 −0.71 −3.931.92E−04 0.069991223 2894790 SYCP2L −0.43 −0.56 −4.20 1.97E−040.070408299 3959451 MYH9 −0.19 −0.25 −4.98 2.07E−04 0.072676061 3954910DERL3 0.30 0.39 3.91 2.25E−04 0.072972083 2844888 BTNL3 −0.31 −0.40−4.65 2.29E−04 0.072972083 2352169 WNT2B −0.32 −0.41 −4.89 2.22E−040.072972083 3351564 PHLDB1 −0.31 −0.40 −3.96 2.21E−04 0.0729720832335014 CYP4Z1 −0.35 −0.45 −4.44 2.14E−04 0.072972083 3041875 OSBPL30.19 0.24 3.82 2.37E−04 0.073973746 2877893 MGC29506 0.48 0.61 3.822.40E−04 0.073973746 3387708 UNQ6228 0.28 0.36 5.65 2.52E−04 0.075088133598758 SMAD6 −0.67 −0.86 −4.45 2.49E−04 0.07508813 2326049 MAN1C1 0.150.19 3.76 2.71E−04 0.075704993 2480168 PRKCE −0.38 −0.49 −4.41 2.92E−040.075704993 3553228 RCOR1 −0.22 −0.28 −3.95 2.69E−04 0.075704993 3159754DMRT2 0.14 0.18 4.61 2.88E−04 0.075704993 2732508 CXCL13 0.90 1.17 4.022.65E−04 0.075704993 2740507 UGT8 0.18 0.24 3.75 2.77E−04 0.0757049933869097 SIGLEC6 0.30 0.38 4.16 2.63E−04 0.075704993 2401193 LUZP1 −0.22−0.29 −4.52 2.90E−04 0.075704993 3834502 CD79A 0.60 0.78 3.90 2.81E−040.075704993 2735459 HERC3 −0.22 −0.29 −4.42 3.56E−04 0.079319593 3161167KIAA1432 −0.20 −0.26 −3.71 3.83E−04 0.079319593 3234760 CUGBP2 −0.20−0.26 −4.76 3.89E−04 0.079319593 3414885 SLC4A8 0.37 0.47 3.93 4.11E−040.079319593 3824427 FAM129C 0.17 0.22 4.54 4.43E−04 0.079319593 2497301TMEM182 0.17 0.22 4.19 4.64E−04 0.079319593 2955999 GPR110 0.69 0.884.21 3.26E−04 0.079319593 2759038 CRMP1 −0.20 −0.25 −3.61 4.29E−040.079319593 2349129 S1PR1 −0.48 −0.62 −4.61 4.53E−04 0.079319593 3372896FOLH1 0.17 0.21 3.82 4.47E−04 0.079319593 3667508 CALB2 0.21 0.28 4.844.14E−04 0.079319593 3404636 GABARAPL1 −0.32 −0.41 −3.72 3.61E−040.079319593 2708066 KLHL6 0.41 0.53 3.85 4.53E−04 0.079319593 7385611HPCAL1 −0.23 −0.30 −4.58 3.44E−04 0.079319593 3633578 CSPG4 −0.31 −0.41−4.68 4.05E−04 0.079319593 3223157 DBC1 −0.65 −0.83 −4.23 3.97E−040.079319593 4001223 RAI2 −0.26 −0.34 −3.82 4.33E−04 0.079319593 3747236C17orf76 0.21 0.27 3.94 3.47E−04 0.079319593 3484895 KL −0.37 −0.48−3.82 3.81E−04 0.079319593 3558418 STXBP6 −0.41 −0.53 −3.95 4.13E−040.079319593 3525313 COL4A1 −0.33 −0.43 −3.73 3.56E−04 0.0793195932411198 TAL1 −0.27 −0.35 −4.07 4.28E−04 0.079319593 2935475 QKI −0.27−0.35 −4.18 3.46E−04 0.079319593 3082874 ARHGEF10 −0.34 −0.44 −4.183.16E−04 0.079319593 2615892 CMTM8 −0.31 −0.39 −4.24 4.67E−040.079319593 3672609 FOXF1 −0.24 −0.31 −3.93 3.94E−04 0.079319593 2599371TMBIM1 −0.18 −0.23 −4.14 3.96E−04 0.079319593 2525533 MAP2 −0.43 −0.55−3.66 4.67E−04 0.079319593 2369339 RALGPS2 0.42 0.54 4.31 4.24E−040.079319593 2664209 SH3BP5 −0.32 −0.41 −4.41 4.07E−04 0.0793195932491788 ATOH8 −0.36 −0.46 −6.31 4.16E−04 0.079319593 2450668 TMEM9 0.170.22 4.59 4.08E−04 0.079319593 2458649 C1orf55 −0.22 −0.29 −3.894.70E−04 0.079319593 2925590 TMEM200A 0.25 0.33 3.73 3.22E−040.079319593 2352106 CTTNBP2NL −0.28 −0.36 −4.34 3.81E−04 0.0793195932343823 LPHN2 −0.48 −0.62 −3.77 4.66E−04 0.079319593 3087703 PDGFRL 0.310.40 3.92 4.61E−04 0.079319593 3467351 ANKS1B 0.20 0.26 3.59 4.77E−040.079687054 2619265 VIPR1 −0.65 −0.84 −3.87 4.91E−04 0.080631196 3822723PKN1 −0.29 −0.37 −4.72 4.96E−04 0.080631196 3933243 PRDM15 0.11 0.144.65 4.94E−04 0.080631196 3235255 ECHDC3 −0.27 −0.35 −4.21 5.12E−040.082469476 3265140 ADRB1 −0.46 −0.59 −4.33 5.28E−04 0.084211404 3438061GPR133 −0.28 −0.35 −4.01 5.47E−04 0.084405378 3780981 KIAA1772 −0.12−0.16 −3.83 5.34E−04 0.084405378 3009959 PTPN12 −0.26 −0.34 −4.055.48E−04 0.084405378 3818842 ZNF358 −0.19 −0.25 −4.38 5.47E−040.084405378 2366798 PRRX1 0.32 0.41 3.60 5.64E−04 0.086141985 2452615SLC45A3 0.31 0.40 4.30 5.69E−04 0.086197425 2891341 IRF4 0.38 0.48 3.796.00E−04 0.090057239 3865998 PNMAL1 0.26 0.33 3.73 6.22E−04 0.0926192073190778 LRRC8A −0.37 −0.48 −4.33 6.42E−04 0.094879887 3975227 MAOA −0.44−0.57 −3.98 6.54E−04 0.095846279 3766533 CD79B 0.16 0.21 4.17 6.84E−040.097822364 2480383 EPAS1 −0.32 −0.41 −3.69 6.81E−04 0.097822364 2615360TGFBR2 −0.15 −0.20 −4.01 6.77E−04 0.097822364 3782069 transcript 0.200.25 4.51 6.90E−04 0.097922646 3782069, GenBank BC012036 2429235 AMPD10.45 0.58 3.47 6.97E−04 0.098051775

TABLE 6 Functional categories enriched among genes associated withregional emphysema severity Direction of Enrichment Category EnrichmentTool h_bcrmolecule: B Cell Receptor Complex Up-regulated DAVID hsa04662:B cell receptor signaling pathway Up-regulated DAVID Immunoglobulindomain Up-regulated DAVID IPR013151: Immunoglobulin Up-regulated DAVIDIPR013783: Immunoglobulin-like fold Up-regulated DAVID signalUp-regulated DAVID SM00409: IG Up-regulated DAVIDHUMORAL_IMMUNE_RESPONSE Up-regulated GSEA GO: 0001525~angiogenesisDown-regulated DAVID GO: 0001568~blood vessel development Down-regulatedDAVID GO: 0001944~vasculature development Down-regulated DAVID GO:0019838~growth factor binding Down-regulated DAVID GO: 0034713~type Itransforming growth factor beta receptor Down-regulated DAVID bindingGO: 0048514~blood vessel morphogenesis Down-regulated DAVIDh_akapCentrosomePathway:Protein Kinase A at the Down-regulated DAVIDCentrosome phosphoprotein Down-regulated DAVID HSA04510_FOCAL_ADHESIONDown-regulated GSEA CXCR4PATHWAY Down-regulated GSEAST_INTEGRIN_SIGNALING_PATHWAY Down-regulated GSEA ECMPATHWAYDown-regulated GSEA HSA04810_REGULATION_OF_ACTIN_CYTOSKELETONDown-regulated GSEA HSA04320_DORSO_VENTRAL_AXIS_FORMATION Down-regulatedGSEA EIF4PATHWAY Down-regulated GSEA GHPATHWAY Down-regulated GSEAREGULATION_OF_CELL_MORPHOGENESIS Down-regulated GSEACYTOSKELETAL_PROTEIN_BINDING Down-regulated GSEA VIPPATHWAYDown-regulated GSEA HSA04520_ADHERENS_JUNCTION Down-regulated GSEASIG_INSULIN_RECEPTOR_PATHWAY_IN_CARDIAC_MYOCYTES Down-regulated GSEAHSA04910_INSULIN_SIGNALING_PATHWAY Down-regulated GSEAINTEGRIN_MEDIATED_CELL_ADHESION_KEGG Down-regulated GSEAINSULIN_RECEPTOR_SIGNALING_PATHWAY Down-regulated GSEA INTEGRINPATHWAYDown-regulated GSEA HSA05214_GLIOMA Down-regulated GSEA GTPASE_ACTIVITYDown-regulated GSEA CELL_MIGRATION Down-regulated GSEAHSA05220_CHRONIC_MYELOID_LEUKEMIA Down-regulated GSEA RUFFLEDown-regulated GSEA LEADING_EDGE Down-regulated GSEAHSA05219_BLADDER_CANCER Down-regulated GSEA VEGFPATHWAY Down-regulatedGSEA RASPATHWAY Down-regulated GSEA HSA05212_PANCREATIC_CANCERDown-regulated GSEA HSA04670_LEUKOCYTE_TRANSENDOTHELIAL_MIGRATIONDown-regulated GSEA SPPAPATHWAY Down-regulated GSEAHSA05131_PATHOGENIC_ESCHERICHIA_COLI_INFECTION_EPEC Down-regulated GSEATDBULIN_BINDING Down-regulated GSEAHSA05130_PATHOGENIC_ESCHERICHIA_COLI_INFECTION_EHEC Down-regulated GSEAHSA05211_RENAL_CELL_CARCINOMA Down-regulated GSEA LIPID_TRANSPORTDown-regulated GSEA HSA03050_PROTEASOME Down-regulated GSEAHSA05215_PROSTATE_CANCER Down-regulated GSEA MITOCHONDRIAL_MEMBRANE_PARTDown-regulated GSEA MTORPATHWAY Down-regulated GSEASIG_PIP3_SIGNALING_IN_CARDIAC_MYOCTES Down-regulated GSEATRANSMEMBRANE_RECEPTOR_PROTEIN_TYROSINE_KINASE_SIGNALING_PATHWAYDown-regulated GSEA REGULATION_OF_CELL_GROWTH Down-regulated GSEAMCALPAINPATHWAY Down-regulated GSEA ERK5PATHWAY Down-regulated GSEAERKPATHWAY Down-regulated GSEA GTPASE_REGULATOR_ACTIVITY Down-regulatedGSEA PTENPATHWAY Down-regulated GSEA PPARAPATHWAY Down-regulated GSEAIGF1RPATHWAY Down-regulated GSEA CCR3PATHWAY Down-regulated GSEAACTIN_BINDING Down-regulated GSEA PDGFPATHWAY Down-regulated GSEAPAR1PATHWAY Down-regulated GSEA PROTEIN_AMINO_ACID_PHOSPHORYLATIONDown-regulated GSEA REGULATION_OF_CELL_MIGRATION Down-regulated GSEAUBIQUITIN_MEDIATED_PROTEOLYSIS Down-regulated GSEA AT1RPATHWAYDown-regulated GSEA ACETYLTRANSFERASE_ACTIVITY Down-regulated GSEAIGF1MTORPATHWAY Down-regulated GSEA RHYTHMIC_PROCESS Down-regulated GSEAPHOSPHORYLATION Down-regulated GSEA SPLICEOSOME Down-regulated GSEAHSA04360_AXON_GUIDANCE Down-regulated GSEA ST_ERK1_ERK2_MAPK_PATHWAYDown-regulated GSEA CORTICAL_CYTOSKELETON Down-regulated GSEAPROTEASOMEPATHWAY Down-regulated GSEA EGFPATHWAY Down-regulated GSEAHSA04720_LONG_TERM_POTENTIATION Down-regulated GSEA PGC1APATHWAYDown-regulated GSEA CIRCADIAN_EXERCISE Down-regulated GSEA NFATPATHWAYDown-regulated GSEA PTDINSPATHWAY Down-regulated GSEAHSA04920_ADIPOCYTOKINE_SIGNALING_PATHWAY Down-regulated GSEAENZYME_LINKED_RECEPTOR_PROTEIN_SIGNALING_PATHWAY Down-regulated GSEAPOST_TRANSLATIONAL_PROTEIN_MODIFICATION Down-regulated GSEAKERATINOCYTEPATHWAY Down-regulated GSEA G_PROTEIN_SIGNALINGDown-regulated GSEA IL2RBPATHWAY Down-regulated GSEAPROTEIN_SERINE_THREONINE_KINASE_ACTIVITY Down-regulated GSEAHISTONE_ACETYLTRANSFERASE_ACTIVITY Down-regulated GSEAREGULATION_OF_ANATOMICAL_STRUCTURE_MORPHOGENESIS Down-regulated GSEATRANSFORMING_GROWTH_FACTOR_BETA_RECEPTOR_SIGNALING_PATHWAYDown-regulated GSEA

Relationship Between Gene Expression Profiles Associated with RegionalEmphysema Severity and COPD-Related Expression Profiles Identified inOther Studies

GSEA was used to examine the relationship between gene expressionprofiles associated with regional emphysema severity as measured by Lmand previously published cross-sectional studies of gene expression inCOPD and COPD-related phenotypes. The genes that decreased in expressionwith increasing emphysema severity were enriched amongst genesdown-regulated in COPD phenotypes from four freely available datasets(FIGS. 3A-3D). In addition, genes that increased in expression withincreasing emphysema severity were enriched amongst genes up-regulatedin COPD phenotypes in three of the same datasets (FIGS. 3A, 3C, and 3D).As an example, the enrichment of genes associated with emphysemaseverity in the data from Golpon et al (American journal of respiratorycell and molecular biology 2004 31:595-600) can be seen in FIG. 4 (thegenes include AMPD1, CD79A, PDGFRL, CD79B, CCR7, UGT8, CD22, PRXCE,MAOA, MAPS2, ACVRL1, STOM, GPR4, S100A8, RCOR1, CUGBP2, TGFBR2, KRT7,TAL1, S1PR1, CSPG4, COL4A2, SMAD6, PKN1, HPCAL1, PECAM1, EDNR8, ENG, andVIPR1). Conversely, expression profiles of genes identified as beingdecreased with COPD in four other cross-sectional studies were enrichedamong the genes that had decreased expression with increasing regionalemphysema severity (FIG. 3E). Expression profiles of genes that werefound to be up-regulated with COPD in Golpon et al (American journal ofrespiratory cell and molecular biology 2004 31:595-600) were alsoenriched in genes that increased in expression with increasing regionalemphysema severity. Contrary to expectation, genes that were found tohave increased expression in COPD phenotypes by Ning et al., (PNAS 2004101:14895-900) and Wang et al., (American journal of respiratory andcritical care medicine 2008 177:402-11) were enriched among genes thatare down-regulated with increasing regional emphysema severity.

Example 3 The Relationship Between Emphysema Severity and TGFβ Signaling

Relationship Between Gene Expression Profiles Associated with RegionalEmphysema Severity and TGFβ-Induced Gene Expression Signatures

Several members of the TGFβ super family were among the genes that haddecreased expression as a function of regional emphysema severity. Thesegenes included ACVRL1, ENG, TGFBR2, and SMAD6. Other components in thisfamily, including BMPR2 (FDR q-value=0.125) and SMAD7 (FDRq-value=0.223), also showed evidence of modest down-regulation. Incontrast, SMAD1 showed evidence of modest up-regulation (FDRq-value=0.101). To determine whether the TGFβ pathway might be affectedby emphysema pathogenesis, previously published studies that hadexamined the effect of TGFβ on gene expression were used to develop acollection of gene expression changes associated with perturbations inTGFβ pathway activity (Malizia, A. P. et al. American journal ofphysiology. Lung cellular and molecular physiology 2008 295:L451-60;Chambers, R. C. et al. The American journal of pathology 2003162:533-46; Classen, S. et al. Journal of immunology 2007 178:6931-40;Koinuma, D. et al. Molecular and cellular biology 2009 29:172-86; Qin,H. et al. BMC systems biology 2009 3:73; Renzoni, E. a et al.Respiratory research 2004 5:24; Verrecchia, F., et al. The Journal ofbiological chemistry 2001 276:17058-62) Genes that exhibitedsignificantly decreased expression with increasing emphysema severitywere enriched in genes that were induced in response to TGFβ treatmentin a total of three datasets (FIGS. 6A-6C).

Similarly, the sets of genes most induced by TGFβ from these threedatasets as well as another four datasets were enriched in genes whoseexpression decreased as a function of emphysema severity (FIG. 6D). Asan example, the enrichment of genes associated with emphysema severityin the data from Malizia et al., (American journal of physiology. Lungcellular and molecular physiology 2008 295:L451-60) is shown in FIG. 5(include the following genes ABTB2, PODXL, EPAS1, HPCAL1, TP8T2, KRT7,COL4A2, COL4A1, NEOD9, NDEL1 and RCOR1). To validate these findings invitro, human lung fibroblasts were cultured with and without TGFβ1 andfound that the set of genes most induced by TGFβ1 were enriched amonggenes that decrease in expression with increasing regional emphysemaseverity (FDR <0.05; FIG. 7A). Immunostaining localized Smad2, a memberof the TGFβ pathway, to the alveolar and airway walls while members ofthe BMP pathway including Smad6 (positive staining) and Smad1 (weakstaining) were primarily seen in vascular endothelial cells (data notshown).

RT-PCR Validation of Gene Expression

Fourteen genes whose expression is significantly correlated withregional emphysema severity or transcription factors that are highlyconnected to these genes in the relevance network (FIGS. 2A-2C) wereselected for RT-PCR validation (Table 7). Three of the subjects withsevere emphysema (6965, 6969, and 6970) were used with four tissue coresper patient. Twelve out of the fourteen genes had significantcorrelation between the expression values derived from the array andRT-PCR (Pearson correlation; p<0.05).

TABLE 7 RT-PCR Validation of 14 genes Gene Direction ANOVA p-valueCD79A* Induced 0.02 CCR7* Induced 0.01 CXCL13* Induced 0.1 BCL11AInduced 0.84 TBX3* Repressed 0.01 SMAD6* Repressed 0.02 S100A8*Repressed 0.09 WFDC1* Repressed 0.05 ACVRL1* Repressed 0.02 FOXF1*Repressed 0.03 GATA2* Repressed 0.09 EPAS1* Repressed 0.06 KLF13*Repressed 0.36 TAL1 Repressed 0.08

The asterisk indicates that the correlation was significant between theexpression values derived from the array and from RT-PCR (Pearson;p<0.05). Column 3 shows the p-value for the correlation of RT-PCRderived expression values to Lm by ANOVA.

Immunohistochemistry for CD79A

In order to investigate whether the up-regulation of components of the Bcell receptor signaling pathway is associated with a change in thevolume of B-cells in lung tissue, the volume fraction (Vv) of CD79Aprotein, a marker for B-cells, was quantified in relation to Lm by IHC.CD79A-positive B cells were observed in the alveolar and small airwaywall tissue by immunohistological staining (data not shown). Vv wasquantified in alveolar tissue for all 64 samples and in small airwaytissue for 43 samples that contained small airways suitable forhistological analysis and was found to be positively correlated to Lm inboth the alveolar and small airway wall tissue (p<0.001), indicatingthat B cell abundance increases as emphysema severity increases.

Example 4 Identification of GHK as a Therapeutic Compound

Connectivity Map

In order to discover potential therapeutic compounds for the treatmentof emphysema, the CMap⁸, a compendium of microarray experiments thatmeasure the effect of many compounds on gene expression in cancer celllines, was employed. Query signatures of genes related to regionalemphysema severity and/or COPD were derived from gene expressionpatterns changing with increasing regional emphysema severity in thisdataset or gene expression patterns correlated to lung functionmeasurements in four other COPD datasets (Golpon, H. A. et al. Americanjournal of respiratory cell and molecular biology 2004 31:595-600;Bhattacharya, S. et al. American journal of respiratory cell andmolecular biology 2009 40:359-67; Spira, A. et al. American journal ofrespiratory cell and molecular biology 2004 31: 601-10; Wang, I.-M. etal. American journal of respiratory and critical care medicine 2008177:402-11). Five additional datasets were used to create querysignatures of gene expression changes associated with TGFβ treatment(Malizia, A. P. et al. American journal of physiology. Lung cellular andmolecular physiology 2008 295:L451-60; Classen, S. et al. Journal ofimmunology 2007 178:6931-40; Koinuma, D. et al. Molecular and cellularbiology 2009 29:172-86; Qin, H. et al. BMC systems biology 2009 3:73;Renzoni, E. a et al. Respiratory research 2004 5:24).

Among the CMap data, gene expression changes resulting from treatmentwith the tripeptide GHK were anti-correlated with expression patternsassociated with increasing regional emphysema severity (p=0.006) and theCOPD-related expression patterns observed in Bhattacharya et al.,(American journal of respiratory cell and molecular biology 200940:359-67) and Goplon et al., (American journal of respiratory cell andmolecular biology 2004 31:595-600). In Spira et al (American journal ofrespiratory cell and molecular biology 2004 31: 601-10), the geneexpression changes induced by GHK were anti-correlated with to thoseassociated with FEV1/FVC (p=0.0002) but positively correlated to thosethat change between cases and controls or with DLCO (p<0.05). Inaddition, GHK-treatment resulted in similar patterns of gene expressionto those observed after TGFβ treatment of cell lines by Malizia et al.,(American journal of physiology. Lung cellular and molecular physiology2008 295:L451-60) (p=0.004).

As the effects of GHK on gene-expression reported in CMap were measuredin cancer cell lines, GHK treatment was verified in human lungfibroblasts. HFL-1 cultures were treated with GHK at two concentrationsor with TGFβ1. Gene expression profiling of these cells demonstratedthat the 200 genes most induced by GHK at 1 μM in cancer cell lines inthe CMap dataset were enriched among genes that increase with treatmentof GHK at 0.1 nM in HFL-1 cultures by GSEA (FIG. 7B). Furthermore, geneswhose expression is decreased with increasing emphysema severity areenriched among genes induced by GHK at 10 nM (data not shown).Similarly, genes that increase after treatment of GHK at eitherconcentration are enriched among genes whose expression decreases withincreasing emphysema (data not shown). Genes whose expression is alteredby GHK treatment at either concentration are also enriched among genesthat change with TGFβ1 treatment (FIGS. 7B-7D and data not shown). Theexpression profile for ITGB1, an integrin important for fibroblastmigration and adhesion, was significantly down-regulated with increasingregional emphysema severity (p=0.0008) in lung tissue and significantlyupregulated with treatment of GHK at 0.1 nM in the HFL-1 (p=0.004). Theprotein levels of ITGB1 were also significantly induced with treatmentof GHK, TGFβ1, or GHK in combination with TGFβ1 suggesting that GHK canmodulate repair processes (FIGS. 8A-8B).

Example 5 Discussion

By measuring gene expression from regions of varying emphysema severitywithin the same lung, the effects of systemic differences betweenindividuals are minimized. Herein, by using a morphologic measurement ofairspace size (Lm) which reflects the degree of alveolar destruction,the gene expression changes observed were specifically related to theemphysematous component of COPD. While HRCT scans are currently thestandard method for grading the severity of emphysema both within andbetween individuals, a close relationship between emphysema as measuredby HRCT scans and Lm as measured by micro-CT has been previouslyreported (Hogg, et al., Proc Am Thorac Soc 2009 6:546-9). The inventors'analysis of 8 specimens per lung representing different degrees ofemphysema from each individual increased the power to detect geneexpression changes associated with regional emphysema severity andrelate these to gene expression differences that have been observedbetween individuals with varying degrees of COPD and/or emphysema.

Identified herein are genes whose expression change as a function ofregional emphysema severity. Herein, the inventors have demonstratedthat progressive emphysematous destruction in COPD is associated withthe down-regulation of genes involved in or downstream of tissueremodeling and wound repair pathways, suggesting a role for defects inECM homeostasis and angiogenesis in the emphysematous destruction thatoccurs in association with chronic inflammation in COPD.

Also provided herein is a compound, GHK, which significantly reversesgene expression patterns associated with increasing emphysema severityand with increasing COPD severity. GHK treatment also induced a patternof gene expression similar to that resulting from TGFβ pathwayactivation. These findings were replicated in human lung fibroblasts. Inaddition, the protein level of β1-integrin was increased with GHKtreatment. Fibroblasts treated with GHK in combination with TGFβ1produced significantly higher levels of β1-integrin compared tofibroblasts treated with TGFβ1 alone (p<0.01).

All references described herein are incorporated herein by reference.

1. An assay for assessing the state of the lungs of a subject, the assaycomprising: subjecting a test sample of a subject to at least oneanalysis to determine the level of expression of at least 2 marker geneproducts from at least 1 of the following 2 groups, Group A, whereinGroup A is selected from the group consisting of: SEQ ID NOs: 1-79,128-205, 253-316, and 379-442 Group B, wherein Group B is selected fromthe group consisting of: SEQ ID NOs: 80-127, 206-252, 317-378, and443-504 wherein an expression profile of 2 or more marker gene productsof Group A which is decreased relative to a reference level and anexpression profile of 2 or more marker gene products of Group B which isincreased relative to a reference level, indicates the presence ofemphysema.
 2. A method comprising: detecting the level of expression ofat least 2 mRNAs selected from at least 1 of the following 2 groups,Group A, wherein Group A is selected from the group consisting of: SEQID NOs: 1-79, 128-205, 253-316, and 379-442. Group B, wherein Group B isselected from the group consisting of: SEQ ID NOs: 80-127, 206-252,317-378, and 443-504; in a sample obtained from a subject.
 3. Th methodof claim 2, wherein the level of expression is detected for at least 2mRNAs selected from each of Group A and Group B.
 4. The method of claim2, wherein the level of expression is detected for at least two mRNAsselected from the group consisting of: SEQ ID NOs: 4; 12; 13; 18; 22;23; 26; 30; 31; 32; 34; 37; 40; 55; 56; 58; 64; 71; 72; 131; 139-140;144; 148-149; 152; 156-158; 160; 163; 166; 181-182; 184; 190; 197-198;256-258; 279; 283-284; 286-287; 382-384; 405; 409-410; and 412-413.4843-1585-6688.1
 5. The method of claim 2, wherein the level ofexpression is detected for at least three mRNAs of Group A and at leastthree mRNAs from Group B.
 6. The method of claim 2, wherein the level ofexpression is detected for mRNAs of at least 2 of the following 6 genes:ITGB1; NEDD9; ACVRL1; SMAD6; TGFBR2; and CD79A.
 7. The method of claim2, wherein the level of expression is detected for mRNAs of at least 3of the following 6 genes: ITGB1; NEDD9; ACVRL1; SMAD6; TGFBR2; andCD79A.
 8. The method of claim 2, wherein the level of expression isdetected for mRNAs of at least 4 of the following 6 genes: ITGB1; NEDD9;ACVRL1; SMAD6; TGFBR2; and CD79A.
 9. The method of claim 2, wherein thelevel of expression is detected for mRNAs of at least 5 of the following6 genes: ITGB1; NEDD9; ACVRL1; SMAD6; TGFBR2; and CD79A.
 10. The methodof claim 2, wherein the level of expression is detected for mRNAs ofITGB1; NEDD9; ACVRL1; SMAD6; TGFBR2; and CD79A.
 11. The method of claim2, wherein the subject is a mammal.
 12. The method of claim 10, whereinthe subject is a human.
 13. The method of claim 2, wherein the subjectis at risk of developing COPD.
 14. The method of claim 2, wherein thesubject smokes tobacco.
 15. The method of claim 2, wherein the subjecthas been exposed to asbestos, air pollution, or environmental hazardssuch as dust, chemicals, fires, and/or smoke.
 16. The method of claim 2,wherein the subject has low levels of α-1 antitrypsin (AAT) in theblood.
 17. The method of claim 2, wherein the subject has been diagnosedwith α-1 antitrypsin (AAT) deficiency.
 18. A method comprising:obtaining a sample from a subject; and detecting the level of expressionof at least 2 gene products selected from at least 1 of the following 2groups, Group A, wherein Group A is selected from the group consistingof: SEQ ID NOs: 1-79, 128-205, 253-316, and 379-442. Group B, whereinGroup B is selected from the group consisting of: SEQ ID NOs: 80-127,206-252, 317-378, and 443-504; in the sample.
 19. An assay foridentifying a compound which can be used in treating COPD and/oremphysema, the assay comprising: using a biological sample containing atleast four marker gene products, wherein at least two of the geneproducts are from the group consisting of SEQ ID NOs: 1-504; measuringthe expression level of the marker genes; comparing the expression levelof the marker genes from the biological sample treated with the compoundto reference levels the marker genes; wherein the reference levels areobtained from a biological sample not treated with the compound; whereinan overall statistically significant difference in the expression levelsof the four marker genes in the biological sample treated with thecompound relative to the reference levels indicates the compound can beused in treating COPD and/or emphysema.